This work was funded by Australian Research Council Discovery Project grant DP180101890 and Rebecca L Cooper Medical Research Foundation project grant PG2018110

A summary of tissue pre-processing steps may be found in Figure 1. All procedures were carried out in compliance with the Animal Care and Ethics Committee of the University of New South Wales in accordance with the guidelines for the use and care of animals for scientific purposes (Australian National Health and Medical Research Council).
1. Sample preparation of fresh frozen brain tissue
<ol>
	<li>Transcardial Perfusion
	<ol>
		<li>Prepare heparinized (2500 U/L) 0.1 M phosphate buffer (PB), pH 7.5. Make dry ice ethanol slurry by mixing dry ice with ethanol. This will have a temperature of approximately &#8722;72 &#176;C and will be used for immediate freezing of the harvested tissue.</li>
		<li>Euthanize adult C57BL/6 and Phox2b-GFP11&#160;(Mouse Genome Informatics database ID MGI:5776545) mice by anesthetizing with sodium pentobarbital (70 mg/kg, i.p.), using a 27.5 inch needle gauge. 
		CAUTION: Pentobarbital is a barbiturate. It is acutely toxic in high doses and may cause death by respiratory arrest. Consult local healthcare, legal and material safety guidelines before use.</li>
		<li>Expose the heart and cannulate the left ventricle with a drawing-up needle (23 inch gauge). Perform transcardial perfusion with heparinized 0.1 M PB&#160;until the blood clears (2-3 minutes) at a flow rate of 11-13 mL/min. Determine blood clearing by monitoring the coloration of the liver and the effusate from the right atrium12.</li>
		<li>Isolate the brain from the skull cavity, immediately embed it in Optimal Cutting Temperature Compound (OCT) in a cryomold or aluminum foil and place it on the dry ice ethanol bath. Store the frozen embedded tissue in an airtight container at - 80 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>Sectioning of fresh frozen tissue
	<ol>
		<li>Set the cryostat temperature to -20 &#176;C. Leave the OCT-embedded tissue and a cryostat chuck in the cryostat for ~30 minutes to allow for equilibration to the new temperature. 
		NOTE: Keep the tissue frozen at all times; transport the tissue from the -80 &#176;C freezer to the cryostat on dry ice.</li>
		<li>Secure the tissue to the pre-chilled cryostat chuck using OCT compound. In this protocol, tissue blocks were mounted onto the chuck in the coronal plane. 
		NOTE: Trim excess OCT from the tissue, using a razor blade, to minimize the amount of OCT being cut by the cryostat and subsequently transferred onto the glass slide.</li>
		<li>Cut 14 &#181;m thick coronal sections and mount them onto charged glass microscopy slides.
		<ol>
			<li>Warm the slides to room temperature before mounting the sections. Once the section has been mounted, keep the slides in a slide box in the cryostat.</li>
			<li>If more than one section needs to be mounted on one slide, warm the area for the second section by placing a finger on the opposite side of the slide for 5-10 seconds to aid adherence of the section to the slide. A cold tissue section will not attach to a cold slide. The sections should adhere to the slides flat; folding will cause them to fall off the slides during wash steps.</li>
			<li>If cracks are noticed in the sections, increase the cryostat temperature by 1-5 &#176;C to avoid this. It is particularly important to place tissue sections in close proximity to one another on the same slide. This will prevent wastage of FISH probes and reagents during the assay.</li>
		</ol>
		</li>
		<li>Store tissue sections mounted onto glass slides in an air-tight container at -80 &#176;C for up to 6 months. 
		NOTE: Keep the sections frozen at all times and avoid freeze thaw cycles, to prevent RNA degradation. Transport the slide box from inside the cryostat to the -80 &#176;C freezer on dry ice.</li>
	</ol>
	</li>
	<li>Fixation of fresh frozen tissue
	<ol>
		<li>On the day the FISH probe assay is to be performed, prepare 4% paraformaldehyde (PFA) in 0.1 M PB, pH 7.5 (4% PFA solution). Filter by passing through filter paper (Grade 1: 11 &#181;m, Table of Materials) in a Buchner funnel or crucible filter. 
		CAUTION : PFA is harmful and toxic by skin contact or inhalation. All procedures with PFA solution should be performed in a fume hood cabinet. PFA solution waste should be disposed of carefully following institutional safety protocols.</li>
		<li>Cool the 4% PFA solution to 4 &#176;C. Transport the slide-mounted tissue from the -80 &#176;C freezer in dry ice and immediately immerse it in the pre-chilled fixative for 15 minutes. 
		NOTE: It is important that this fixation step does not exceed 15 minutes as over-fixation will result in non-specific background labelling.</li>
	</ol>
	</li>
	<li>Dehydration of fresh frozen tissue
	<ol>
		<li>Dehydrate tissue sections by submerging the slides in graded concentrations of ethanol. In a Coplin jar, first submerge in 50%, then 70% and finally absolute ethanol, for 5 minutes each at room temperature. Repeat the final absolute ethanol incubation a second time.</li>
		<li>Air dry slides and, outline the group of sections using a hydrophobic barrier pen, ensuring that the internal area is kept to a minimum. 
		NOTE: Make sure that the glass slide is completely dry before drawing the hydrophobic barrier. The hydrophobic barrier should surround the tissue sections completely without gaps and must be dry before further processing.</li>
	</ol>
	</li>
</ol>
2. Sample preparation of fixed frozen brain tissue
<ol>
	<li>Transcardial perfusion fixation
	<ol>
		<li>Euthanize mice by anesthetizing with sodium pentobarbital (70 mg/kg, i.p) followed by transcardial perfusion, first with 0.1 M PB then 4% PFA solution. Fix with 10 minutes of perfusion at 11-13 mL/min.</li>
		<li>Isolate the brain from the skull cavity following perfusion-fixation and submerge overnight in 4% PFA solution, at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Tissue sectioning of fixed tissue
	<ol>
		<li>Rinse the brain in sterile 0.1 M phosphate buffered saline (PBS) before removing the meningeal layers, with the aid of a dissecting microscope, using fine forceps.</li>
		<li>Cut the brain precisely into blocks (separate the brainstem from the forebrain prior to vibratome sectioning) using a brain matrix (Table of Materials). Specifically, cut the brainstem caudally at the pyramidal decussation and dissect away the cerebellum. Similarly, cut the forebrain immediately rostral to the optic chiasm.</li>
		<li>Secure the tissue onto a vibrating microtome chuck using cyanoacrylate and embed in 2% agar solution.</li>
		<li>Cut 30 &#181;m thick tissue sections using a vibrating microtome and store cut sections in cryoprotectant solution (30% RNase free sucrose, 30% ethylene glycol, 1% polyvinylpyrrolidone (PVP-40),&#160;in 0.1 M PB, pH 7.4). Tissue sections may be stored in cryoprotectant at -20 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Preparation of fixed sections prior to FISH
	<ol>
		<li>On the day of FISH, wash free floating sections three times, for 10 minutes per wash, to remove the cryoprotectant solution. To wash, place sections in 0.1 M PBS in a 12 well cell culture plate and agitate on a rotating platform shaker (90 - 100 rpm).</li>
		<li>After washes, use a paintbrush to mount sections on glass microscopy slides and air dry for at least 2 hours. 
		NOTE: The sections should adhere flat onto the slides as any pronounced folds will cause them to detach during washes.</li>
		<li>Using a hydrophobic barrier pen, draw a barrier around the sections to restrict the FISH reagents to the sections. Once more, it is important to minimize the internal area of the outline drawn with the barrier pen. 
		&#8203;POSSIBLE BREAK POINT: The sections could be stored at room temperature, overnight, to continue the assay the next day.</li>
	</ol>
	</li>
</ol>
3. FISH assay
NOTE: The rest of the protocol applies to both fresh frozen and fixed frozen tissue.
<ol>
	<li>Prepare the reagents and instruments for hybridization and amplification steps.
	<ol>
		<li>Set a benchtop incubator and water bath to 40 &#176;C.</li>
		<li>Prepare a humidified, light-protected chamber for incubating slides. Humidification prevents drying of tissues - slides are securely located above a moist reservoir. Ideally, the chamber is made of heavy-duty polystyrene, it is light-proof and airtight to maintain a saturated water vapor atmosphere. Closure of the chamber relies on minimal friction to avoid movement. We used a slide box lined with wet laboratory wipes (Table of Materials) at the bottom. Place the slide box inside the incubator to prewarm it to 40 &#176;C.</li>
		<li>Warm the 50x Wash Buffer (Table of Materials) and probes to 40 &#176;C for 10 minutes, using the water bath, then cool to room temperature.</li>
		<li>Prepare 1 L of 1x Wash Buffer from the 50x stock concentration.</li>
		<li>Prepare probe mixture (Table of Materials): the C1 probe is ready to use at stock concentration whereas C2 and C3 probes are shipped as 50x concentration and require dilution with the diluent supplied in the kit. 
		NOTE: Probe mixtures can be stored at 4 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Protease treatment
	<ol>
		<li>Incubate sections with Protease III (Table of Materials) at room temperature for 30 minutes. 
		NOTE: Ensure that Protease III and incubation reagents in downstream processes (probe mixture, amplification solutions, blocking buffer and antibody sera) cover the sections entirely. A pipet tip may be used to spread the reagent onto the section to cover the entire area inside the hydrophobic barrier.</li>
		<li>Wash slides twice with 0.1 M PBS, for 2 min each time, in&#160;a large plastic square Petri dish. Here a 245 mm x 245 mm square bioassay dish was used (Table of Materials). Hold from one side of the dish and tilt gently 3-5 times. After washes, flick excess 0.1 M PBS from slide and immediately add the next reagent. Do not let tissue sections dry. 
		NOTE: During each wash, the slides are immersed in solution at room temperature. This is the workflow for all subsequent wash steps. The fixed 30 &#181;m thick sections dislodge from slides more easily than 14 &#181;m thick sections, be gentle during the washes.</li>
	</ol>
	</li>
	<li>Hybridization and amplification
	<ol>
		<li>After washing off the protease solution, place the slides in the humidified, prewarmed chamber. Incubate sections with probe mixture (Table of Materials) for 2 hours at 40 &#176;C inside a benchtop incubator. 
		&#8203;NOTE: Ensure there are at least 2 sections set aside for positive and negative control probes to assess sample RNA quality and optimal permeabilization. Positive control probes target house-keeping genes; here, these were a&#160;cocktail of RNAs targeting ubiquitin C (UBC; high-abundance), peptidylpropyl isomerase B (PPIB; moderate-abundance) and RNA polymerase 2a (POLR2A; low-abundance). Negative control probes target the bacterial 4-hydroxy-tetrahydrodipicolinate reductase (DapB) gene, which is normally absent in mouse brain samples. Positive DapB signal indicates non-specific signal and/or bacterial contamination of the sample.</li>
		<li>Following hybridization with the probe mixture, the signal amplification steps consist of incubation with Amp 1-FL (30 minutes), then with Amp 2-FL (15 minutes), followed by Amp 3-FL (30 minutes) and finally Amp 4-FL (15 minutes) - each at 40 &#176;C. Using the dropper bottles provided, cover tissue sections with amplification solution. Proceed to IHC assay following the last amplification step.</li>
		<li>Rinse slides with Wash Buffer twice for 2 minutes between probe hybridization and each amplification step.</li>
	</ol>
	</li>
</ol>
4. IHC Assay
<ol>
	<li>IHC blocking step
	<ol>
		<li>To prevent non-specific binding of antibodies, incubate the sections for 1 h at room temperature with blocking solution containing 10% normal horse serum, 0.3% Tween20 in 1x TBSm (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% merthiolate) following the FISH assay. Prepare primary antibodies in a dilution buffer containing 1x TBSm, 5% normal horse serum and 0.1% Tween20. Primary antibody suppliers are listed in the Table of Materials.</li>
	</ol>
	</li>
	<li>Immunohistochemistry
	<ol>
		<li>Remove excess blocking buffer by flicking the slide and incubate sections with primary antibodies overnight at 4 &#176;C.</li>
		<li>Wash slides 3 times (5 minutes each) with 1x TBSm and incubate with secondary antibody in diluent containing 1x TBSm, 1% normal horse serum and 0.1% Tween20 for 2 hours at room temperature. Secondary antibodies used in this protocol are listed in the Table of Materials.</li>
		<li>Wash slides 3 times with 1x TBSm (5 minutes each) before coverslipping with mounting medium with or without DAPI (Table of Materials).</li>
	</ol>
	</li>
</ol>
5. Imaging
<ol>
	<li>Examine immunostaining under an epifluorescence microscope equipped with a camera (see Table of Material for details). Acquire representative images at 20x magnification and save as TIFF files.</li>
	<li>Export representative images into an image processing software (Table of Materials) for brightness/contrast adjustment to increase the clarity and to reflect true rendering.</li>
</ol>
6. OPTIONAL: Quantitative analysis of target transcripts
NOTE: This is a methods article and quantitative results are not provided. The method of quantification presented here is sourced from Dereli et al.10.
<ol>
	<li>Acquire images from the regions of interest as explained in 5.1 and apply the same microscope and camera settings (such as exposure time and light intensity) to all images of the same fluorophore.</li>
	<li>Plot the neuronal profiles using an image analysis software (Table of Materials).</li>
	<li>Align the sections with reference to Bregma level according to a stereotaxic brain atlas13.</li>
	<li>Apply the same brightness and contrast to all the images of the same fluorophore. Only consider the neurons with DAPI-stained nuclei.</li>
	<li>Manually count the number of mRNA, protein expressing, mRNA/mRNA, protein/protein and mRNA/protein coexpressing cells within the region of interest.</li>
	<li>To decrease bias in the experimental results, have the person quantifying experimental outcomes blinded to the experimental groups.</li>
	<li>Apply Abercrombie correction14 to total cell counts by using the following Abercombie equation: 
	Corrected cell count = manual cell count x section thickness / (section thickness + nuclear size) 
	For example, for 14 &#181;m thick sections, the average nuclear width is calculated to be 7.7 &#177; 0.3 &#181;m and average section thickness is 14 &#177; 1 &#181;m based on 30 cells and 10 sections respectively in 5 animals10. According to the Abercrombie equation, corrected cell count would be manual cell count x 14/(14+7.7).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61709/61709fig01.jpg" /> 
Figure 1: Parallel workflow of tissue pre-processing steps for both fresh-frozen and paraformaldehyde fixed tissue. Processing steps for fresh-frozen tissue are displayed in the red outlined boxes, whereas those for paraformaldehyde (PFA) fixed tissue are displayed in the blue outlined boxes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61709/61709fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61709/61709fig02.jpg" /> 
Figure 2: Summary of combined FISH probe and immunohistochemistry procedure. Following tissue pre-processing, the slide mounted tissue is encircled using a hydrophobic barrier pen, as seen in the first frame, and incubated in a protease solution at room temperature. Following washes, tissue is transferred to a benchtop incubator for hybridization for 2 hours before sequential amplification steps. The in situ hybridization system utilizes a proprietary &#39;Z probe&#39; design, preamplifiers and amplifiers as seen in frames 3-66. Once tissue has undergone FISH probe processing, it is washed before blocking with normal horse serum. The primary antibody incubation is carried out overnight at 4 &#176;C to maximize antibody-antigen binding. The secondary antibody incubation (2 hours) was carried out at room temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61709/61709fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14 (1), 22-29 (2012).</li><li>Annese, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope+dual+ISH-IHC+technology+to+study+angiogenesis+in+diffuse+large+B-cell+lymphomas.">RNAscope dual ISH-IHC technology to study angiogenesis in diffuse large B-cell lymphomas.</a> Histochemistry and Cell Biology. 153 (3), 185-192 (2020).</li><li>Morrison, J. A., McKinney, M. C., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+in+vivo+gene+expression+during+collective+cell+migration+using+an+integrated+RNAscope,+immunohistochemistry+and+tissue+clearing+method.">Resolving in vivo gene expression during collective cell migration using an integrated RNAscope, immunohistochemistry and tissue clearing method.</a> Mechanisms of Development. 148, 100-106 (2017).</li><li>Gross-Thebing, T., Paksa, A., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+detection+of+multiple+transcripts+combined+with+localization+of+proteins+in+whole-mount+embryos.">Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos.</a> BMC Biology. 12, 55 (2014).</li><li>Stempel, A. J., Morgans, C. W., Stout, J. T., Appukuttan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+and+cell-specific+confirmation+of+RNA+and+protein+in+the+mouse+retina.">Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina.</a> Molecular Vision. 20, 1366-1373 (2014).</li><li>Kersigo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+RNAscope+whole+mount+approach+that+can+be+combined+with+immunofluorescence+to+quantify+differential+distribution+of+mRNA.">A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA.</a> Cell and Tissue Research. 374 (2), 251-262 (2018).</li><li>Grabinski, T. M., Kneynsberg, A., Manfredsson, F. P., Kanaan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+combining+RNAscope+in+situ+hybridization+with+immunohistochemistry+in+thick+free-floating+brain+sections+and+primary+neuronal+cultures.">A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.</a> PLoS One. 10 (3), 0120120 (2015).</li><li>Baleriola, J., Jean, Y., Troy, C., Hengst, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+axonally+localized+mRNAs+in+brain+sections+using+high-resolution+in+situ+hybridization.">Detection of axonally localized mRNAs in brain sections using high-resolution in situ hybridization.</a> Journal of Visualized Experiments. (100), e52799 (2015).</li><li>Fe Lanfranco, M., Loane, D. J., Mocchetti, I., Burns, M. P., Villapol, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+fluorescent+in+situ+hybridization+(FISH)+and+immunofluorescence+imaging+for+detection+of+cytokine+expression+in+microglia/macrophage+cells.">Combination of fluorescent in situ hybridization (FISH) and immunofluorescence imaging for detection of cytokine expression in microglia/macrophage cells.</a> Bio-Protocol. 7 (22), (2017).</li><li>Dereli, A. S., Yaseen, Z., Carrive, P., Kumar, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+respiratory-related+brain+regions+to+long-term+hypercapnia:+focus+on+neuropeptides+in+the+RTN.">Adaptation of respiratory-related brain regions to long-term hypercapnia: focus on neuropeptides in the RTN.</a> Frontiers in Neuroscience. 13, 1343 (2019).</li><li>Lazarenko, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensitivity+and+ultrastructure+of+the+retrotrapezoid+nucleus+in+Phox2b-EGFP+transgenic+mice.">Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice.</a> Journal of Comparative Neurology. 517 (1), 69-86 (2009).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Paxinos, G., Franklin, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2004).</li><li>Abercrombie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+nuclear+population+from+microtome+sections.">Estimation of nuclear population from microtome sections.</a> Anatomical Records. 94, 239-247 (1946).</li><li>Kerr, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+knock-in+mice+expressing+fluorescently+tagged+galanin+receptors+1+and+2.">The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2.</a> Molecular and Cellular Neurosciences. 68, 258-271 (2015).</li><li>Kachidian, P., Pickel, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+tyrosine+hydroxylase+in+neuronal+targets+and+efferents+of+the+area+postrema+in+the+nucleus+tractus+solitarii+of+the+rat.">Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat.</a> Journal of Comparative Neurology. 329 (3), 337-353 (1993).</li><li>Stornetta, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Phox2b+by+brainstem+neurons+involved+in+chemosensory+integration+in+the+adult+rat.">Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat.</a> Journal of Neuroscience. 26 (40), 10305-10314 (2006).</li><li>Gilmor, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+putative+vesicular+acetylcholine+transporter+in+rat+brain+and+localization+in+cholinergic+synaptic+vesicles.">Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles.</a> Journal of Neuroscience. 16 (7), 2179-2190 (1996).</li><li>Fisher, J. M., Sossin, W., Newcomb, R., Scheller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+neuropeptides+derived+from+a+common+precursor+are+differentially+packaged+and+transported.">Multiple neuropeptides derived from a common precursor are differentially packaged and transported.</a> Cell. 54 (6), 813-822 (1988).</li><li>Towle, A. C., Lauder, J. M., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+tyrosine-hydroxylase+immunocytochemistry+in+paraffin+sections+using+pretreatment+with+proteolytic-enzymes.">Optimization of tyrosine-hydroxylase immunocytochemistry in paraffin sections using pretreatment with proteolytic-enzymes.</a> Journal of Histochemistry and Cytochemistry. 32 (7), 766-770 (1984).</li><li>Biancardi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+excitatory,+inhibitory,+and+modulatory+afferent+projections+to+the+anatomically+defined+active+expiratory+oscillator+in+adult+male+rats.">Mapping of the excitatory, inhibitory, and modulatory afferent projections to the anatomically defined active expiratory oscillator in adult male rats.</a> Journal of Comparative Neurology. 529 (4), 853-884 (2021).</li><li>Matthews, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+in+the+brainstem:+an+excitatory+disynaptic+pathway+for+control+of+whisking.">Feedback in the brainstem: an excitatory disynaptic pathway for control of whisking.</a> Journal of Comparative Neurology. 523 (6), 921-942 (2015).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+methods+of+immunohistochemistry.">Principles and methods of immunohistochemistry.</a> Methods in Molecular Biology. 1641, 115-128 (2017).</li><li>Shi, S. R., Key, M. E., Kalra, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+in+formalin-fixed,+paraffin-embedded+tissues:+an+enhancement+method+for+immunohistochemical+staining+based+on+microwave+oven+heating+of+tissue+sections.">Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.</a> Journal of Histochemistry and Cytochemistry. 39 (6), 741-748 (1991).</li><li>Yamashita, S., Katsumata, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+in+immunohistochemistry:+mechanisms+and+applications.">Heat-induced antigen retrieval in immunohistochemistry: mechanisms and applications.</a> Methods in Molecular Biology. 1560, 147-161 (2017).</li><li>Yamashita, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+heat-induced+antigen+retrieval:+analyses+in+vitro+employing+SDS-PAGE+and+immunohistochemistry.">Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry.</a> Journal of Histochemistry and Cytochemistry. 53 (1), 13-21 (2005).</li><li>Yamashita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval:+mechanisms+and+application+to+histochemistry.">Heat-induced antigen retrieval: mechanisms and application to histochemistry.</a> Progress in Histochemistry and Cytochemistry. 41 (3), 141-200 (2007).</li></ol>

In the neurosciences, FISH and IHC are routinely used to investigate the spatial organization and functional significance of mRNA or proteins within neuronal subpopulations. The protocol described in this study enhances the capacity for simultaneous detection of mRNAs and proteins in brain sections. Our combined multiplex FISH-IHC assay enabled phenotypic identification of distinct neuronal subpopulations in the NTS in both fresh frozen and fixed brain preparations. FISH-IHC in fixed frozen tissue preparations produced reliable IHC outcomes. For example, multiplex FISH for low and high abundance mRNAs (GalR1 and GlyT2 respectively) and IHC (targeting tyrosine hydroxylase) revealed that GalR1 and GlyT2 are expressed in non-catecholaminergic NTS neurons. IHC for TH was not successful in fresh frozen tissue, highlighting the limited capacity for FISH-IHC in fresh frozen preparations.
ISH may be more appropriate than IHC in a range of scenarios. First, IHC may not perform well when detecting low abundance proteins, such as receptors. Using ISH to target relatively higher abundance mRNAs for these proteins improves detectability1. Second, proteins such as neuropeptides are often trafficked to the axonal terminals following translation in the cell soma19. When neuropeptides are targeted with IHC, the axonal processes and terminals of cells label with the antibody, but not the soma, reducing the capacity to identify the cell of origin or perform quantitative analysis of the number of cells. However, since mRNAs which code for all proteins are found localized to the soma, the ISH technique is advantageous. Finally, antibodies are not readily available for some protein species, or the available antibodies are appropriate for other proteomics techniques (e.g., western blot) but not IHC. In these circumstances, mRNA labelling methods prove useful. A caveat is that mRNAs may not always be translated into protein, and so they only provide a proxy for protein identification. Since commercial FISH kits can be costly and the ISH probes are less likely to be commercially available compared to antibodies, combining FISH with IHC presents a cost and time effective strategy for increasing the number of targets that can be labelled simultaneously.
Fresh frozen versus fixed tissue preparation was a factor conferring successful IHC following FISH probe assay. We tested IHC using antibodies targeting nuclear, vesicular and cytoplasmic proteins and found reliable labelling of membrane-bound proteins (vAChT and Phox2b) on fresh frozen samples but not cytoplasmic proteins (TH and GFP). Coexpression of Phox2b protein and mRNA with &#39;flocculent&#39; Phox2b-GFP labelling validated that neurons expressing the transcript also expressed the related protein, confirming&#160;the neurochemical identity of the neurons (Figure 5). In contrast, fixed frozen tissue preparations yielded reliable IHC labelling regardless of subcellular localization of the antigen. Previous studies have demonstrated that protease (e.g., pronase8,20) pretreatment can have a detrimental effect on IHC. The contents of the protease solution utilized in the RNAscope protocol is proprietary, and permeabilization by protease is recommended for RNAscope probe access into cells. Labelling of cytoplasmic proteins using the antibodies described here has been previously verified on free floating 30 &#181;m fixed frozen mouse brain sections10,21,22. We slide-mounted 30 &#181;m thick fixed samples and performed the FISH-IHC protocol, as opposed to the 14 &#181;m thick fresh frozen sections recommended by the manufacturer. In the absence of assay modifications, or alteration of other&#160;variables (antigen retrieval, higher antibody concentration, change of protease), reliable IHC was achieved on thick, fixed samples with demonstrated labelling of the cytoplasm and axonal processes together with FISH probe labelling (Figure 6). While similar approaches were employed by other research groups7,8,9, the current study achieved combined ISH-IHC on neurons and in a fluorescent set-up.
There were a series of critical steps in the methods to take note of. For the fresh frozen preparation, fixation time should not exceed 15 minutes; longer fixation times elicited higher background labelling. The protease step was optimized since tissues of different thickness and from various organs require different types of protease to achieve permeabilization. Fixed frozen sections adhere less to glass slides and dislodge more easily during wash steps. Hence, extra care must be taken in manual handling of fixed frozen sections, to avoid tissue loss or damage.
Although we found combined FISH and IHC to be an effective strategy, the disadvantages include cost and technically demanding assay when combining the two methods. One limitation of the study is that a side-by-side comparison of the two tissue preparation protocols was not performed. Also, our evaluation of results was limited by the number of channels the epifluorescent microscope could accommodate; the set-up allowed a maximum of 4 channels at a given time: 346, 488, 550 and 647 nm (excitation light). We were able to achieve multiplex labelling of 5 targets by labelling two proteins with different subcellular localizations using the same flurophore (Figure 4, Table 1). By using a confocal microscope, discrete excitation of many additional fluorophores may be used for individual protein labelling via IHC, or for imaging of fluorescent molecules expressed by transgenes.
Combined FISH and fluorescent IHC can reduce the reliability of each technique in isolation. In the future, we aim to improve cytoplasmic protein labelling on fresh frozen tissue&#160;with an antigen retrieval treatment23. Previous studies show that heat induced antigen retrieval increases accessibility of the protein epitope24,25,26. Heat treatment cleaves the crosslinks and methylol groups of the protein and unfolds the antigens in tissues, exposing epitopes which would otherwise be hidden in the tertiary protein structure under biological conditions. This accessibility may improve the success of protein labelling26,27. Additionally, we will target different epitopes of the same cytoplasmic protein to determine if the success of protein-antibody labelling depends on the specific antibody clones used.
In conclusion, combined FISH and IHC is useful for neurochemical identification of heterogenous populations of cells in the brain, such as those in the NTS. This study presents two protocols assaying different mouse brainstem tissue preparations - fresh frozen, or fixed - for simultaneous multiplex fluorescent labelling of mRNA and proteins in situ. Both protocols may be widely applied to detect the expression pattern of low abundance mRNAs, such as GalR1. Thick (30 &#181;m) fixed frozen preparations permeabilized with protease conferred more reliable cytoplasmic protein detection and more tissue handling challenges, when&#160;compared to thin (14 &#181;m) fresh frozen preparations.

Proteins and mRNAs that neurochemically define subpopulations of neurons are commonly identified with a combination of immunohistochemistry (IHC) and/or in situ hybridization (ISH), respectively. Combining ISH with IHC techniques facilitates the characterization of colocalization patterns unique to functional neurons (neurochemical coding) by maximizing multiplex labelling capacity.
Fluorescent ISH (FISH) methods, including RNAscope, have higher sensitivity and specificity compared to earlier RNA detection methods such as radioactive ISH and non-radioactive chromogenic ISH. FISH enables visualization of single mRNA transcripts as punctate stained spots1. Furthermore, the RNAscope assay allows an increased number of RNA targets to be labeled at a time, using different fluorophore tags. Despite these advantages, technical limitations may affect the number of fluorophores/chromogens that can be used in a single experiment. These include availability of microscope filter sets; such considerations are compounded when neurochemical identification uses combined FISH and IHC, compared to using each technique in isolation, since inherent steps optimal for one method may be detrimental to the other.
Previous application of FISH combined with IHC has demonstrated the expression of specific cellular targets in human B-cell lymphomas2, chick embryos3, zebrafish embryos4, mouse retina5 and mouse inner ear cells6. In these studies, tissue preparation was either formalin-fixed paraffin embedded (FFPE)2,3,5 or fresh whole mount4,6. Other studies applied chromogenic RNAscope on fixed mouse and rat brain preparations7,8,9. In particular, Baleriola et al.8 described two different tissue preparations for combined ISH-IHC; fixed mouse brain sections and FFPE human brain sections. In a recent publication, we combined FISH and fluorescent IHC on fresh frozen sections, to simultaneously visualize low abundance mRNA (galanin receptor 1, GalR1), high abundance mRNA (glycine transporter 2, GlyT2) and vesicular acetylcholine transporter (vAChT) protein10 in the brainstem reticular formation.
The nucleus of the solitary tract (NTS) is a major brain region involved in autonomic function. Located in the hindbrain, this heterogeneous population of neurons receives and integrates a vast number of autonomic signals, including those that regulate breathing. The NTS harbors several neuronal populations, which may be phenotypically characterized by the expression pattern of mRNA targets including GalR1 and GlyT2 and protein markers for the enzyme tyrosine hydroxylase (TH) and the transcription factor Paired-like homeobox 2b (Phox2b).
The RNAscope proprietor recommends fresh frozen tissue preparations, but tissue prepared by whole animal transcardial perfusion fixation, along with long term cryoprotection (storage at -20 &#176;C) of fixed frozen tissue sections, is common in many laboratories. Hence, we sought to establish protocols for FISH in combination with IHC using fresh frozen and fixed frozen tissue preparations. Here, we provide for fresh frozen and fixed frozen brain sections: (1) a protocol for combined FISH and fluorescent IHC (2) a description of the quality of mRNA and protein labelling produced, when utilizing each preparation (3) a description of the expression of GalR1 and GlyT2 in the NTS.
Our study revealed that, when combined with RNAscope methodology, IHC success varied in fresh frozen and fixed frozen preparations and, was dependent upon localization of the target proteins within the cell. In our hands, membrane bound protein labelling was always successful. In contrast, IHC for cytoplasmic protein required troubleshooting even in cases where the cytoplasmic protein was overexpressed in a transgenic animal (Phox2b-GFP)11. Finally, while GalR1 is expressed in non-catecholaminergic neurons in the NTS, GlyT2 expression is absent in the NTS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ANIMALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mouse</td>
			<td>Australian BioResources, Moss Vale</td>
			<td>MGI: 2159769</td>
			<td></td>
		</tr>
		<tr>
			<td>Phox2b-eGFP mouse</td>
			<td>Australian BioResources, Moss Vale</td>
			<td>MGI: 5776545</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate</td>
			<td>Loctite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-Sodium</td>
			<td>Clifford Hallam Healthcare</td>
			<td>1070760</td>
			<td>Consult local veterinary supplier or pharmacy.</td>
		</tr>
		<tr>
			<td>Lethabarb (Sodium Pentabarbitol) Euthanasia Injection</td>
			<td>Virbac (Australia) Pty Ltd</td>
			<td>N/A</td>
			<td>Consult a veterinarian for local pharmaceutical regulations regarding Sodium Pentabarbitol</td>
		</tr>
		<tr>
			<td>Molecular grade agarose powder</td>
			<td>Sigma Aldrich</td>
			<td>5077</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound, 118mL</td>
			<td>Scigen Ltd</td>
			<td>4586</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, prilled, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>441244-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone, average mol wt 40,000&#160; (PVP-40)</td>
			<td>Sigma-Aldrich</td>
			<td>PVP40</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>With or without DAPI</td>
		</tr>
		<tr>
			<td>RNAscope Multiplex Fluorescent Reagent Kit (up to 3-plex capability)</td>
			<td>Advanced Cell Diagnostics, Inc. (ACD Bio)</td>
			<td>ADV320850</td>
			<td>Includes 50x Wash buffer and Protease III</td>
		</tr>
		<tr>
			<td>RNase Away</td>
			<td>Thermo-Fisher Scientific</td>
			<td>7003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Sigma-Aldrich</td>
			<td>252859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20, for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop incubator</td>
			<td>Thermoline scientific micro incubator</td>
			<td>Model: TEI-13G</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Matrix, Mouse, 30g Adult, Coronal, 1mm</td>
			<td>Ted Pella</td>
			<td>15050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Drawing-up needle (23 inch gauge)</td>
			<td>BD</td>
			<td>0288U07</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Barrier Pen</td>
			<td>Vector labs</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes Delicate Task Wipes</td>
			<td>Kimberley Clark Professional</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX51</td>
			<td>Olympus</td>
			<td>BX-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Coleparmer Masterflex</td>
			<td>L/S Series&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Retiga 2000R Digital Camera</td>
			<td>QImaging</td>
			<td>RET-2000R-F-CLR</td>
			<td>colour camera</td>
		</tr>
		<tr>
			<td>SuperFrost Plus Glass Slides (White)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating Microtome (Vibratome)</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper, Grade 1, 110 mm diameter</td>
			<td>Merck</td>
			<td>WHA1001110</td>
			<td></td>
		</tr>
		<tr>
			<td>SOFTWARES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CorelDRAW&#160;</td>
			<td>Corel Corporation</td>
			<td>Version 7</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI (ImageJ Distribution)</td>
			<td>Open Source/GNU General Public Licence (GPL)</td>
			<td>N/A</td>
			<td>ImageJ 2.x: Rueden, C. T.; Schindelin, J. &#38; Hiner, M. C. et al. (2017), &#34;ImageJ2: ImageJ for the next generation of scientific image data&#34;, BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-z&#160;&#160; and Fiji: Schindelin, J.; Arganda-Carreras, I. &#38; Frise, E. et al. (2012), &#34;Fiji: an open-source platform for biological-image analysis&#34;, Nature methods 9(7): 676-682, PMID 22743772, doi:10.1038/nmeth.2019&#160;</td>
		</tr>
		<tr>
			<td>PRIMARY ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase Antibody</td>
			<td>Millipore Sigma</td>
			<td>AB1542</td>
			<td>Sheep polyclonal (1:1000 dilution), RRID: AB_90755</td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase Antibody, clone LNC1</td>
			<td>Millipore Sigma</td>
			<td>MAB318</td>
			<td>Mouse monoclonal (1:1000 dilution), RRID: AB_2201528</td>
		</tr>
		<tr>
			<td>Anti-Vesicular Acetylcholine Transporter (VAchT) Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>ABN100</td>
			<td>Goat polyclonal (1:1000 dilution), RRID: AB_2630394</td>
		</tr>
		<tr>
			<td>GFP Antibody</td>
			<td>Novus Biologicals</td>
			<td>NB600-308</td>
			<td>Rabbit polyclonal (1:1000 dilution), RRID: AB_10003058</td>
		</tr>
		<tr>
			<td>Phox2b Antibody (B-11)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376997</td>
			<td>Mouse monoclonal (1:1000 dilution), RRID: AB_2813765</td>
		</tr>
		<tr>
			<td>SECONDARY ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-545-152</td>
			<td>Donkey anti-Rabbit (1:400 dilution), RRID: AB_2313584</td>
		</tr>
		<tr>
			<td>AMCA AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>713-155-147</td>
			<td>Donkey anti-Sheep (1:400 dilution), RRID: AB_AB_2340725</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Goat IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>705-175-147</td>
			<td>Donkey anti-Goat (1:400 dilution), RRID: AB_2340415</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-175-151</td>
			<td>Donkey anti-Mouse (1:400 dilution), RRID: AB_2619678</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Sheep IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)</td>
			<td>Jackson ImmunoResearch</td>
			<td>713-175-147</td>
			<td>Donkey anti-Sheep (1:400 dilution), RRID: AB_2340730</td>
		</tr>
		<tr>
			<td>RNASCOPE PROBES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Galanin Receptor 1 oligonucleotide probe</td>
			<td>ACDBio</td>
			<td>448821-C1</td>
			<td>targets bp 482 - 1669 (Genebank ref: NM_008082.2)</td>
		</tr>
		<tr>
			<td>Glycine transporter 2 oligonucleotide probe</td>
			<td>ACDBio</td>
			<td>409741-C3</td>
			<td>targets bp 925 - 2153 (Genebank ref: NM_148931.3)</td>
		</tr>
		<tr>
			<td>Phox2b oligonucleotide probe</td>
			<td>ACDBio</td>
			<td>407861-C2</td>
			<td>targets bp 1617 - 2790 (Genebank ref: NM_008888.3)</td>
		</tr>
	</tbody>
</table>

combining multiplex fluorescence in situ hybridization with fluorescent immunohistochemistry on fresh frozen or fixed mouse brain sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Here, we outline a method for combining multiplex FISH with fluorescent IHC to localize mRNA expression for GalR1 and GlyT2 using fresh-frozen and paraformaldehyde fixed tissues respectively in the mouse NTS. A pipeline of the tissue processing, FISH and IHC procedures described in the methods is displayed in Figure 1 and Figure 2. Table 1 provides a summary of the FISH probe and antibody combinations used in each figure.
Control probes are routinely assayed concurrently with target probe, to ensure integrity of the workflow and confirm sample quality. The absence of DapB labelling confirms sound tissue quality and integrity, and the absence of bacterial contamination (Figure 3A). Labelling from positive control probes targeting ubiquitin C (UBC, high abundance), peptidylpropyl isomerase B (PPIB, moderate abundance) and RNA polymerase 2a (POLR2A, low abundance) mRNA confirms RNA integrity and the signal observed between assays may be used to calibrate inter assay variability (Figure 3B). To validate FISH probe expression, we used control tissues that have been previously described to express the mRNA transcript. For example, GalR1 mRNA expression, was confirmed to be positive in the thalamus as previously described10,15. Phox2b mRNA distribution was additionally verified by colabelling with Phox2b antibody; we confirmed that FISH labelling was present only in neurons that were also positively stained using the Phox2b antibody (Figure 5).
To distinguish GalR1+ neurons in the NTS from neighbouring nuclei, we used additional neurochemical markers. TH, Phox2b or Phox2b-GFP immunoreactivity (Figure 4-6), and Phox2b FISH (Figure 5 and Figure 6) differentiated the NTS from other nuclei in the dorsal brainstem as NTS neurons have previously been reported to express Phox2b and TH16,17. Since the NTS is nestled by cholinergic nuclei - it lies dorsal to the hypoglossal nucleus and dorsal motor nucleus of the vagus (DMNX), and ventral to the vestibular nucleus - we co-labelled with the cholinergic marker vAChT18 (Figure 4). Therefore, the expression of GalR1 within the NTS was assessed in relation to TH and Phox2b, whilst vAChT labelling aided spatial orientation with respect to rostrocaudal, dorsoventral and mediolateral coordinates. We found all TH immunoreactive and GalR1 mRNA positive neurons in the NTS were Phox2b-GFP immunoreactive, but not all Phox2b-GFP immunoreactive neurons in the NTS were TH immunoreactive or GalR1 mRNA positive (Figure 4). Also, we demonstrated that mRNA for the low abundance receptor GalR1 was absent in TH and vAChT immunoreactive neurons.
In fresh frozen preparations, when combined with the FISH probe assay, IHC success was dependent on subcellular location of the target protein. For example, vAChT (a synaptic vesicle membrane-bound protein) was clearly immunolabelled, whereas TH and GFP (cytoplasmic proteins) were indefinitely immunolabelled and only faintly observed (Figure 4). We describe this indefinite labelling as &#39;flocculent&#39; because cells lacked a clear outline and proved difficult to distinguish from the background. On the same fresh frozen tissue section, GalR1 FISH probe labelling of cytoplasmic GalR1 mRNA was punctate and clearly observed (Figure 4).
Furthermore, since the TH and vAChT antibodies are raised in the same host, both proteins were labelled using the same secondary antibody and therefore the same color fluorophore (excitation light: 594). They are easily distinguished for two reasons: they never co-label in the same neurons, and the subcellular localisation is different for these proteins; vAChT in vesicles exhibiting a punctate appearance, and TH in the cytoplasm and neuronal processes.
To support our hypothesis that IHC quality (in fresh frozen preparations) is dependent on protein subcellular localisation, we compared labelling for Phox2b mRNA (located in the cytoplasm), GFP (over-expressed in cytoplasm) and Phox2b protein (primarily found in the nucleus) in neurons. As expected, our results show overlap of Phox2b mRNA, GFP and Phox2b antibody labelling in individual neurons of the NTS (Figure 5). Cells with cytoplasmic mRNA labelling corresponded with cells exhibiting nuclear labelling of the Phox2b protein providing validation of the combined FISH-IHC method. Although cytoplasmic Phox2b-GFP had a flocculent appearance, nuclear Phox2b protein signal was clear and specific. In conclusion, when combined with FISH on fresh frozen preparations, membrane-bound proteins including vAChT and Phox2B exhibit higher quality immunolabelling than cytoplasmic proteins.
In contrast, IHC was reliable irrespective of subcellular localization, when performed on fixed frozen sections in combination with FISH. Multiplex FISH for GlyT2 mRNA and Phox2b mRNA was successful, as shown in Figure 6. GlyT2 mRNA positive neurons were located ventral to the NTS and not within the NTS. GlyT2+ and Phox2b+ neurons did not colocalize. A subpopulation of Phox2b+ NTS neurons was TH immunoreactive and none contained GlyT2 mRNA. TH immunoreactive neurons are apparent on the same tissue section, exhibiting positively labelled soma and neuronal processes (Figure 6). This contrasts with the &#39;flocculent&#39; appearance of TH immunoreactive neurons in fresh frozen tissue sections. Thus, the fixed frozen preparation described here is an alternative method of tissue preparation which enables reliable targeting of cytoplasmic proteins immunohistochemically, in combination with RNAscope.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61709/61709fig03.jpg" /> 
Figure 3:&#160;Representative microscopic images from coronal mouse forebrain sections at the level of the lateral septum (Bregma 1.1 to -0.1) showing labelling of positive and negative control probes. (A) A&#160;lack of signal following ISH with bacterial 4-hydroxy-tetrahydrodipicolinate reductase (DapB) confirms the absence of background signals. (B) Labelling with positive control probes targeting ubiquitin C (UBC), peptidylpropyl isomerase B (PPIB) and RNA polymerase 2a (POLR2A) illustrates the signal to be expected from high, moderate and low abundance targets respectively. Scale bars are 50 &#181;m. All images were acquired with 20x objective. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61709/61709fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61709/61709fig04.jpg" /> 
Figure 4: Representative microscopic images of a fresh frozen coronal brainstem section from a Phox2b-GFP mouse showing combined labelling of GalR1 mRNA (FISH) and 3 proteins (IHC) in the nucleus of the solitary tract (NTS) region. Insets in A are enlarged in B. GalR1 mRNA is indicated by punctate FISH probe labelling (arrowheads). Antibodies targeting the cytoplasmic proteins GFP and tyrosine hydroxylase (TH) exhibited &#34;flocculent&#34; labelling (arrows). Vesicular acetylcholine transporter (vAChT) immunoreactivity is demonstrated (red punctate labelling) in the hypoglossal nucleus (XII). Scale bars are 100 &#181;m in A and 25 &#181;m in B. All images were acquired with 20x objective. Other abbreviations: area postrema (AP), medial vestibular nucleus (MVe). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61709/61709fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61709/61709fig05.jpg" /> 
Figure 5:&#160;Representative microscopic images of a fresh frozen coronal brainstem section from a Phox2b-GFP mouse, illustrating targeting of Phox2b in the nucleus of the solitary tract (NTS) with three different approaches: Phox2b mRNA (FISH), GFP (IHC) and Phox2b protein (IHC). Phox2b protein is localized to the nucleus. Insets in A are enlarged in B. Arrows indicate neurons that are triple labelled with Phox2b probe (orange-550), GFP antibody (green-488) and Phox2b antibody (red-647). Scale bars are 100 &#181;m in A and 25 &#181;m in B. All images are acquired with a 20x objective. Other abbreviations: area postrema (AP). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61709/61709fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61709/61709fig06.jpg" /> 
Figure 6: Representative images from fixed frozen coronal brainstem sections demonstrating successful FISH combined with reliable immunolabelling of cytoplasmic proteins (tyrosine hydroxylase [TH]). Double FISH showing glycine transporter 2 (GlyT2-red-647, filled arrowheads) and Phox2b (yellow-550, arrows) mRNA labelling in nucleus of the solitary tract (NTS) region. FISH was combined with IHC for TH protein (blue-346, empty arrowheads). Insets in A are enlarged in B. Scale bars are 25 &#181;m. All images were acquired with a 20x objective. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61709/61709fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Primary antibody or RNAscope probe</td>
			<td>Secondary antibody or 
			Amp 4-FL-Alt Display module</td>
			<td>Excitation (nm)</td>
			<td>Tissue preparation</td>
		</tr>
		<tr>
			<td>Figure 3</td>
			<td>probe</td>
			<td>POLR2A (C1)</td>
			<td>Amp 4-FL-Alt B Display module</td>
			<td>647</td>
			<td rowspan="4">fresh frozen</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>PPIB (C2)</td>
			<td>Amp 4-FL-Alt B Display module</td>
			<td>488</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>UBC (C3)</td>
			<td>Amp 4-FL-Alt B Display module</td>
			<td>550</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>DapB (C1, C2, C3)</td>
			<td>Amp 4-FL-Alt B Display module</td>
			<td>647, 488, 550</td>
		</tr>
		<tr>
			<td></td>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>346</td>
			<td></td>
		</tr>
		<tr>
			<td>Figure 4</td>
			<td>antibody</td>
			<td>rabbit-anti-GFP&#8239;</td>
			<td>donkey-anti-rabbit</td>
			<td>488</td>
			<td rowspan="5">fresh frozen</td>
		</tr>
		<tr>
			<td></td>
			<td>antibody</td>
			<td>sheep-anti-TH</td>
			<td>donkey-anti-sheep</td>
			<td>647</td>
		</tr>
		<tr>
			<td></td>
			<td>antibody</td>
			<td>goat-anti-vAChT</td>
			<td>donkey-anti-goat</td>
			<td>647</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>GalR1 (C1)</td>
			<td>Amp 4-FL-Alt B Display module</td>
			<td>550</td>
		</tr>
		<tr>
			<td></td>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>346</td>
		</tr>
		<tr>
			<td>Figure 5</td>
			<td>antibody</td>
			<td>rabbit-anti-GFP&#8239;</td>
			<td>donkey-anti-rabbit</td>
			<td>488</td>
			<td rowspan="4">fresh frozen</td>
		</tr>
		<tr>
			<td></td>
			<td>antibody</td>
			<td>mouse-anti-Phox2b</td>
			<td>donkey-anti-mouse</td>
			<td>647</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>Phox2b (C2)</td>
			<td>Amp 4-FL-Alt A Display module</td>
			<td>550</td>
		</tr>
		<tr>
			<td></td>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>346</td>
		</tr>
		<tr>
			<td>Figure 6</td>
			<td>antibody</td>
			<td>mouse-anti-TH</td>
			<td>donkey-anti-mouse</td>
			<td>346</td>
			<td rowspan="3">fixed</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>GlyT2</td>
			<td>Amp 4-FL-Alt A Display module</td>
			<td>647</td>
		</tr>
		<tr>
			<td></td>
			<td>probe</td>
			<td>Phox2b</td>
			<td>Amp 4-FL-Alt A Display module</td>
			<td>550</td>
		</tr>
	</tbody>
</table>
Table 1: FISH probe, antibody and corresponding flurophore combinations used in Figures 3-6.

Watch this Scientific Journal Video about Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections at JoVE.com

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

combining-multiplex-fluorescence-situ-hybridization-with-fluorescent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.6K Views.  University of New South Wales. This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Video: Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.