We would like to thank Athanasios Papangelis, for critical comments on the manuscript. FM is supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/M009424/1). IP is supported by the American Heart Association Scientist Development Grant (17SDG33060002).

Mouse heart tissue samples for this study were obtained in compliance with the relevant laws and institutional guidelines and were approved by Yale University Institutional Animal Care and Use Committee.
1. Solution Preparation
<ol>
	<li>Prepare RNase free ddH2O, by treating 5 L of ddH2O with 5 mL of 0.1% diethylpyrocarbonate (DEPC) overnight (O/N), at room temperature (RT). Autoclave (121&#160;&#176;C) to deactivate the DEPC. Use DEPC-treated ddH2O for the preparation of downstream solutions as indicated. 
	CAUTION: DEPC is a known carcinogen, only handle in the fume hood.</li>
	<li>Prepare 1x Phosphate Buffered Saline (PBS) solution by dissolving 5 PBS tablets in 1 L of DEPC-treated ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Prepare 0.1% non-ionic detergent/PBS (PBST) by diluting 1 mL of non-ionic detergent per 1 L of 1x PBS.</li>
	<li>Prepare 90%, 70%, 50% and 30% ethanol in DEPC-treated ddH2O.</li>
	<li>Prepare a 10 mg/mL stock solution of Proteinase K (ProK) in DEPC-treated ddH2O. Aliquot and store at -20&#160;&#176;C. Prepare a 20 &#956;g/mL working solution of ProK by diluting 100 &#956;L of ProK stock solution in 50 mL of DEPC-treated ddH2O. Make fresh before use.</li>
	<li>Prepare 4% paraformaldehyde (PFA) by adding 2 g of PFA in 50 mL of 1x PBS. Heat at 65&#160;&#176;C with occasional shaking, until dissolved. Aliquot and store at -20&#160;&#176;C. 
	Caution: PFA is a known carcinogen, only handle in the fume hood.</li>
	<li>Prepare a 3 M NaCl solution by adding 87.8 g to 500 mL of ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Prepare a 1 M MgCl2 solution by adding 20.3 g to 100 mL of ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Prepare 1 M Tris pH 8.0 by dissolving 121.1 g in 800 mL of ddH2O. Adjust the pH to 8.0 with a few drops of 1 N HCl, bring the volume to 1 L and autoclave (121&#160;&#176;C). Prepare a working solution of 50 mM Tris pH 8.0 by diluting 2.5 mL of 1 M Tris pH 8.0 in 47.5 mL of ddH2O.</li>
	<li>Prepare 1 M Tris pH 9.5 by dissolving 60.6 g in 400 mL of ddH2O. Adjust the pH to 9.5 with a few drops of 1 N HCl, bring the volume to 500 mL and autoclave (121&#160;&#176;C).</li>
	<li>Prepare 0.13 M 1-Methylimidazole pH 8.0 by diluting 1.6 mL of 1-Methylimidazole to 130 mL of DEPC-treated ddH2O. Adjust the pH to 8.0 with approximately 450 &#181;L of 12 N HCl. Add 16 mL of 3 M NaCl and bring the volume to 160 mL. Make fresh before use. 
	Caution: 1-Methylimidazole is harmful to mucous membranes, eyes, and skin, only handle in the fume hood.</li>
	<li>Prepare 0.16 M N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC) by diluting 176 &#181;L of EDC to 10 mL of 0.13 M 1-Methylimidazole. Adjust the pH to 8.0 with approximately 100 &#181;L of 12 N HCl. Make fresh before use. 
	CAUTION: EDC is harmful to mucous membranes, eyes, and skin, only handle in the fume hood.</li>
	<li>Prepare 1% H2O2 by diluting 1.5 mL of 30% H2O2 in 50 mL of 1x PBS. Make fresh before use.</li>
	<li>Prepare 20x SSC pH 7.0 by dissolving 175.3 g of NaCl and 88.2 g of sodium citrate (Na3C6H5O7) in 800 mL of ddH2O. Adjust the pH to 7.0 with a few drops of 1 N HCl, bring the volume to 1 L and autoclave (121&#160;&#176;C).
	<ol>
		<li>Prepare a working solution of 2x SSC pH 7.0 by diluting 20 mL of 20x SSC pH 7.0 in 180 mL of ddH2O.</li>
		<li>Prepare a working solution of 1x SSC pH 7.0 by diluting 2.5 mL of 20x SSC pH 7.0 in 47.5 mL of ddH2O.</li>
		<li>Prepare a working solution of 0.2x SSC pH 7.0 by diluting 5 mL of 2x SSC pH 7.0 in 45 mL of ddH2O.</li>
	</ol>
	</li>
	<li>Prepare 1x hybridization solution by diluting 0.5 mL of 2x microRNA ISH buffer in 0.5 mL of DEPC-treated ddH2O. Make fresh before use.</li>
	<li>Prepare 200 mM stock solution of Levamisole by adding 250 mg to 5 mL of ddH2O. Aliquot and store at -20&#160;&#176;C.</li>
	<li>Prepare blocking solution for step 5 (10% sheep serum/1% BSA/0.2 mM Levamisol) by diluting 1 mL of sheep serum in 9 mL PBST, and adding 0.1 g of BSA. Add 10 &#956;L of Levamisole before using. Make fresh before use.</li>
	<li>Prepare blocking solution for step 6 (10% goat serum/1% BSA) by diluting 1 mL of goat serum in 9 mL of PBST, and adding 0.1 g of BSA. Store at 4&#160;&#176;C and use within a week.</li>
	<li>Prepare prestaining solution by diluting 3.3 mL of 3 M NaCl, 5 mL of 1 M MgCl2,10 mL of 1 M Tris pH 9.5, 100 &#181;L of Tween-20, 100 &#181;L of Levamisole in 81.5 mL of ddH2O. Make fresh before use.</li>
	<li>Use nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indoly phosphate (BCIP) tablets to prepare 20 mL of Alkaline Phosphatase (AP) substrate solution according to the manufacturer&#39;s instructions. Add 20 &#956;L of Levamisole. Make fresh before use and protect from light.</li>
	<li>Prepare KTBT solution by adding 4 g of Tris, 4.4 g of NaCl and 375 mg of KCl in 500 mL of ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Optional: Prepare a DAPI working solution (1 &#956;g/mL) by diluting 50 &#956;L of DAPI stock solution (1 mg/mL) in 50 mL of 1x PBS. 
	NOTE: Standard solutions such as 3 M NaCl, 1 M MgCl2, 1 M Tris-HCl, nuclease-Free ddH2O, 1x PBS and 20x SSC can be alternatively purchased. For this protocol, 50 mL glass Coplin jars or 20 mL polypropylene slide mailer jars have been used.</li>
</ol>
2. Tissue Preparation
NOTE: The mouse heart sections used here were prepared by Yale Pathology Tissue Services, from Formalin-Fixed/Paraffin-embedded tissue, cut at 5 &#956;m and positioned on charged slides.
<ol>
	<li>Tissue rehydration.
	<ol>
		<li>Warm the slides at 60&#160;&#176;C for 10 min, in the hybridization oven until paraffin is visibly melting.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of commercially available clearing agent (see Table of Materials) for 10 min, twice.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of 100% ethanol for 2 min, twice, followed by 90% ethanol for 2 min, 70% ethanol for 2 min, twice, 50% ethanol for 2 min and 30% ethanol for 2 min.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of DEPC-treated ddH2O for 5 min.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min.</li>
	</ol>
	</li>
	<li>Tissue de-proteinating.
	<ol>
		<li>Incubate slides in a glass Coplin jar containing 50 mL of 20 &#956;g/mL ProK working solution, at 37&#160;&#176;C for 15 min in the hybridization oven. 
		CAUTION: Under-digestion may result in poor or no signal development, while over-digestion may result in tissue degradation and development of background staining. Optimization may be required for this step (1&#8211;20 &#956;g/mL ProK, 5&#8211;20 min incubation, RT-37&#160;&#176;C).</li>
		<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min, at RT, twice.</li>
	</ol>
	</li>
	<li>Tissue fixation.
	<ol>
		<li>Use a hydrophobic pen to draw a water repellent circle around the tissue on each slide.</li>
		<li>Apply 500 &#181;L of 4% PFA solution to the length of each slide and incubate slides horizontally for 10 min, at RT.</li>
		<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min, at RT, twice.</li>
		<li>Wash the slides in a glass Coplin jar containing 50 mL of 0.13 M 1-methylimidazole for 10 min, at RT, twice.</li>
		<li>Apply 500 &#181;L of 0.16 M EDC solution to the length of each slide and incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
		<li>Rinse the slides in a glass Coplin jar containing 50 mL of 50 mM Tris pH 8.0 at RT.</li>
		<li>Rinse the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT, twice. 
		NOTE: Both PFA and EDC fixation steps are required for miRNA crosslinking and should not be omitted.31</li>
	</ol>
	</li>
	<li>Endogenous peroxidase block.
	<ol>
		<li>Incubate slides in a glass Coplin jar containing 50 mL of 1% H2O2 for 30 min at RT.</li>
		<li>Rinse the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT, twice.</li>
	</ol>
	</li>
</ol>
3. Hybridization
<ol>
	<li>Preheat hybridization solution (500 &#181;L per slide) at 50&#160;&#176;C in the hybridization oven, until the target temperature, is reached (10&#8211;15 min).</li>
	<li>Prepare the hybridization chamber by placing 2 pieces of filter or tissue paper in the bottom of the chamber and wetting the paper with 50% formamide/1x SSC. 
	Caution: Formamide is harmful to mucous membranes, eyes and skin, only handle in the fume hood.</li>
	<li>Apply 200 &#181;L of hybridization solution to the length of each slide. Incubate slides horizontally for 1&#8211;3 h at 50&#160;&#176;C, in a humidified chamber.</li>
	<li>Prepare the probe solution by mixing 0.5 &#181;L stock probe (25 &#181;M) in 250 &#181;L ofhybridization solution (final concentration 50 nM) in a 1.5 mL tube. 
	NOTE: Different miRNAs are expressed at different levels, so optimization regarding the probe concentration may be required for this step (1&#8211;50 nM), to avoid no signal or high background development.</li>
	<li>Apply 250 &#181;L of probe solution to the length of each slide and place an RNase free coverslip on top. Avoid the formation of bubbles.</li>
	<li>Carefully seal the hybridization chamber with parafilm and incubate O/N at 50&#160;&#176;C. 
	NOTE: The hybridization temperature should be set approximately 30&#160;&#176;C below the RNA melting temperature (Tm) of each probe. Optimization may be required. Here, 50&#160;&#176;C is used as the hybridization/wash temperature for miRNA-182, adjust accordingly for different probes.</li>
</ol>
4. Stringency Washes
<ol>
	<li>Prewarm 200 mL of 2x SSC at 50&#160;&#176;C in the hybridization oven, until the target temperature is reached (20&#8211;40 min).</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 2x SSC at 50&#160;&#176;C for 5 min, or until the coverslips loosen.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 2x SSC at RT for 5 min, twice.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 0.2x SSC at RT for 5 min.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of PBST at RT for 5 min.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min. 
	NOTE: Use of RNase-free conditions is no longer necessary since the RNA-RNA hybrids are considered stable.</li>
</ol>
5. DIG Antibody Detection
<ol>
	<li>Use a hydrophobic pen to re-draw a water repellent circle around the tissue on each slide if necessary.</li>
	<li>Apply 500 &#181;L of blocking solution to the length of each slide. Incubate slides horizontally for 30 min at RT, in a humidified chamber.</li>
	<li>Prepare the DIG antibody solution (1:1,000) by diluting 0.5 &#956;L of DIG antibody in 500 &#181;L ofblocking solution in a 1.5 mL tube. 
	CAUTION: Optimization may be required for this step (1:500&#8211;1:2,000).</li>
	<li>Apply 500 &#181;L of DIG antibody solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of PBST at RT for 5 min, thrice.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of prestaining solution at RT for 5 min, twice.</li>
	<li>Incubate slides in a polypropylene slide mailer jar containing 20 mL ofAP substrate solution at 37&#160;&#176;C, for 6&#8211;24 h, protected from light. 
	Caution: Color development should be checked every 2 h. Optimization may be required for this step.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of KTBT solution at RT for 5 min, twice.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min.</li>
</ol>
6. cTnT Antibody Detection
<ol>
	<li>Use a hydrophobic pen to re-draw a water repellent circle around the tissue on each slide if necessary.</li>
	<li>Apply 500 &#181;L of blocking solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
	<li>Prepare the cTnT antibody solution (1:100) by diluting 2 &#956;L of cTnT antibody in 200 &#956;L ofblocking solution in a 1.5 mL tube.</li>
	<li>Apply 200 &#181;L of cTnT antibody solution to the length of each slide. Incubate slides horizontally O/N at 4&#160;&#176;C, in a humidified chamber.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, thrice.</li>
	<li>Prepare the anti-rabbit-568 antibody solution (1:500) by diluting 0.5 &#956;L of anti-rabbit-568 antibody in 250 &#956;L ofblocking solution in a 1.5 mL tube.</li>
	<li>Apply 250 &#181;L of anti-rabbit-568 antibody solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, thrice.</li>
	<li>Optional: Apply 250 &#181;L of DAPI working solution to the length of each slide, and incubate slides horizontally for 1 min at RT, protected from light.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, twice.</li>
</ol>
7. Mounting and Imaging
<ol>
	<li>Mount the slides with 2&#8211;3 drops of mounting medium and a glass cover slip. Allow the slides to dry flat, O/N, at 4&#160;&#176;C, protected from light.</li>
	<li>Proceed to epifluorescent or confocal microscopy.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+ventricular+remodeling:+mechanisms:+part+1+of+2.">Pathological ventricular remodeling: mechanisms: part 1 of 2.</a> Circulation. 128, 388-400 (2013).</li><li>Benjamin, E. 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=Heart+Disease+and+Stroke+Statistics-2017+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association.</a> Circulation. 135, e146-e603 (2017).</li><li>Pena, J. 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=miRNA+in+situ+hybridization+in+formaldehyde+and+EDC-fixed+tissues.">miRNA in situ hybridization in formaldehyde and EDC-fixed tissues.</a> Nature Methods. 6, 139-141 (2009).</li></ol>

The authors have nothing to disclose.

miRNA transcript detection can be achieved through different techniques that vary in sensitivity, specificity and quantitative power. Here, we demonstrate the coupling of miRNA in situ hybridization with immunostaining and describe a protocol that allows for concurrent assessment of the expression levels of miRNA and protein molecules, on the same heart sections. We first show how to perform in situ hybridization of DIG-labeled LNA miRNA probes on paraffin embedded heart sections. Next, we describe how to perform immunostaining for cTnT on the same sections. Finally, we demonstrate how to merge the resulting color and fluorescent images. This protocol has been optimized for Formalin Fixed (4 &#176;C, O/N)/Paraffin embedded mouse heart tissues, with the use of DIG-labeled LNA miRNA probes and cTnT. Further optimization may be required for different tissues, probe or antibody types, as outlined below.
RNase-free conditions should be carefully implemented throughout the steps that lead up to probe hybridization, as RNase contamination can severely compromise the outcome of the experiment. All solutions should be made with DEPC-treated ddH2O, and all Coplin jars should be treated with an RNase decontamination solution. Additionally, the following considerations should be taken into account when working with different DIG-labeled LNA miRNA probes. The hybridization temperature depends on the melting temperature (Tm) of each probe. As a general rule, a temperature that is 30 &#176;C below the given RNA Tm or 20 &#176;C below the given DNA Tm should be used for probe hybridization. Furthermore, the ideal probe concentration should be optimized. It usually lies between 1&#8211;50 nM. Lastly, the probe containing hybridization solution may be heated to 95 &#176;C for 5 min to denature any secondary structures, if this is a concern. An important step to test the specificity of a probe, especially when used for the first time, is to include a negative (scrambled) as well as positive (for example U6) control probe, to ensure experimental success (Figure 2). Similar controls (for example a CD31 endothelial antibody) should be used during the immunostaining step.
A common problem in in situ hybridization experiments is the development of staining artifact/background signal. To avoid or minimize artifact staining steps, the tissue should be protected from drying out throughout all steps, specifically during long incubations. We recommend covering the slides with RNase-free coverslips during the hybridization step, particularly when high temperatures are used, and the use of humidifying chambers. We also suggest the use of a 568 or 594 fluorophore to detect cTnT or the protein of interest during the immunostaining step. This will eliminate any autofluorescence that often arises with the use of 488 fluorophores used on formaldehyde fixed tissues.
Another variable we recommend paying attention to is the duration of AP staining development, which depends on the levels of the specific miRNA present in the tissue. We suggest checking AP staining progression every 2 h, for the first experiment. Further optimization may include changing the temperature of incubation (RT-37 &#176;C) parameter. Lastly, if background begins to develop before the true miRNA probe staining, or if the AP staining solution changes color to blue-brown, we suggest washing the slides in PBST twice, and replacing the staining solution with a freshly made one.
Finally, although this protocol has been optimized for Formalin-Fixed/Paraffin-embedded mouse heart tissues, it may be adapted for alternatively processed tissues (PFA fixed/OCT embedded) and/or the use of the different probe or antibody types. A limitation of combining in situ hybridization and immunostaining that should be considered is the failure of several antibodies to bind to their respective epitopes, due to the possible destruction of the latter ones at the high temperatures used during the hybridization and stringency washing steps. Further limitations of both in situ hybridization and immunostaining techniques most often refer to the power of these techniques to detect very low concentrations of miRNA or protein molecules respectively. Signal amplification kits are available to use for either DIG or antibody detection when miRNA or protein abundance is a concern. Such kits also provide a fluorescence alternative for the detection of miRNAs, which, when coupled with fluorescent immunostaining can simplify image acquisition. Alternative methods, such as Real-Time PCR and Western Blotting that have greater sensitivity, can also address low abundance issues. However, contrary to in situ hybridization/immunostaining protocol, these techniques provide no information on the tissue-specific localization of the miRNA/protein molecules.
To summarize, we describe a protocol that provides simultaneous detection of miRNA and protein molecules on the same tissue, which we believe will be an invaluable tool in miRNA research on mouse models.

micro-RNAs (miRNAs) are short (18&#8211;25 nucleotides), single-stranded, noncoding RNAs that function as negative regulators of gene expression at the post-transcriptional level by inhibiting messenger RNA (mRNA) translation and/or promoting mRNA degradation1. miRNAs are transcribed from introns or exons of coding or noncoding genes and are cleaved in the nucleus by DROSHA, to precursor miRNAs (pre-miRNAs), which are short stem-loop structures of 70 nucleotides2. Following cytoplasmic export, pre-miRNAs are further processed by DICER into mature miRNAs that span 18&#8211;25 nucleotides3,4. Subsequently, the RNA-induced silencing complex (RISC) incorporates these miRNAs as single-stranded RNAs, which allows for their binding to the 3&#39; untranslated region (3&#39;-UTR) of their target mRNAs to suppress their expression3,5.
Within the last three decades, since they were first identified, miRNAs have emerged to master&#160;regulators of gene expression, whose own expression levels are tightly controlled6. Roles for miRNAs have been described in organ development7,8,9,10,11,12, maintenance of homeostasis13,14, as well as disease contexts that include neurological15,16,17,18,19, cardiovascular20, autoimmune conditions21,22, cancers23,24, and others25. The increasing appreciation for the relevance of miRNA expression patterns has brought forward the need for reliable detection methods of miRNA transcripts. Such methods include Real Time PCR, microarrays, Northern Blotting, in situ hybridization and others, which vary in the sensitivity, specificity, and quantitative power, predominantly due to the fact that miRNA transcripts are comprised of short and highly homologous sequences6.
We recently reported an important role for miRNA-182 in the development of the myocardial hypertrophy26, a condition describing the structural adaptation of the heart in response to elevated hemodynamic demands27,28. Cardiac hypertrophy is characterized by the increase in the myocardial mass, which, if associated with maladaptive remodeling29, can lead to increased risk for heart failure, a condition accounting for 8.5% of all deaths attributable to cardiovascular disease30.
Here, we describe our protocol that combines in situ hybridization with a digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe and immunostaining for the concurrent detection of miRNA and protein molecules on mouse heart tissue sections, in our model of cardiac hypertrophy.

<table>
	<tbody>
		<tr>
			<td>Diethylpyrocarbonate</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>DEPC</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>American Bioanalytical</td>
			<td>AB02038</td>
			<td>non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Histoclear</td>
			<td>National Diagnostics</td>
			<td>HS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR Grade</td>
			<td>Sigma Aldrich</td>
			<td>3115879001</td>
			<td>ProK</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>PFA</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher</td>
			<td>S271</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>ThermoFisher</td>
			<td>M33</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma Aldrich</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Solution, 1 N</td>
			<td>ThermoFisher</td>
			<td>SA48</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Solution, 12 N</td>
			<td>ThermoFisher</td>
			<td>S25358</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>1-Methylimidazole</td>
			<td>Sigma Aldrich</td>
			<td>336092</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>39391</td>
			<td>EDC</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution H2O2</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td>H2O2</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>S1804</td>
			<td>Sodium Citrate</td>
		</tr>
		<tr>
			<td>miRCURY LNA miRNA ISH Buffer Set (FFPE)</td>
			<td>Qiagen</td>
			<td>339450</td>
			<td>scramble miRNA/U6 snRNA</td>
		</tr>
		<tr>
			<td>miRCURY LNA mmu-miR-182 detection probe</td>
			<td>QIagen</td>
			<td>YD00615701</td>
			<td>5&#39;-DIG and 3&#39;-DIG labelled</td>
		</tr>
		<tr>
			<td>Levamisol hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>31742</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9647</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>NBT/BCIP Tablets</td>
			<td>Sigma Aldrich</td>
			<td>11697471001</td>
			<td>NBT-BCIP</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>ThermoFisher</td>
			<td>P217</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>DAPI solution (1mg/ml)</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>ThermoFisher</td>
			<td>12-545E</td>
			<td>Glass coverslip</td>
		</tr>
		<tr>
			<td>Plastic coverslip</td>
			<td>Grace Bio-Labs</td>
			<td>HS40 22mmX40mmX0.25mm</td>
			<td>RNA-ase free plastic coverslip</td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP, Fab fragments</td>
			<td>Sigma Aldrich</td>
			<td>11093274910</td>
			<td>DIG antibody</td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td>Pap pen</td>
		</tr>
		<tr>
			<td>Anti-Cardiac Troponin T antibody</td>
			<td>Abcam</td>
			<td>ab92546</td>
			<td>cTnT antibody</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>ThermoFisher</td>
			<td>A-11011</td>
			<td>anti-rabbit-568 antibody</td>
		</tr>
		<tr>
			<td>Dako Fluorescence Mounting Medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma Aldrich</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized Formamide</td>
			<td>American Bioanalytical</td>
			<td>AB00600</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>ThermoFisher</td>
			<td>UVP HB-1000 Hybridizer</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue-specific mirna expression profiling in mouse heart sections using in situ hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

miRNA in situ hybridization was optimized on mouse heart sections using a scramble miRNA and U6-snRNA, which served as negative and positive controls respectively. Positive staining is indicated in blue, while the negative staining is indicated by the lack of color development (Figure 1A-1B). Cardiomyocyte specific expression of miRNA-182 was assessed in heart sections from control and PlGF overexpressing mice. The mice carrying the PlGF transgene under a &#945;MHC promoter develop cardiac hypertrophy, secondary to increased angiogenesis, within 6 weeks of transgene activation26. We performed in situ hybridization and found increased expression of miRNA-182 in the hearts of PlGF mice, compared to controls (Figure 2A-2H), indicated by the blue staining. To determine which cell types express miRNA-182, we then performed immunostaining for cTnT on the same sections. We found miRNA-182 co-localized with the cTnT-positive cardiomyocyte cells, as well as DAPI stained nuclei, in both control and PlGF mouse heart sections (Figure 2C-2H), indicated by the red and blue staining respectively. These images were with a 20X objective.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57920/57920fig1.jpg" /> 
Figure 1: Negative and Positive in situ hybridization staining. (A) In situ hybridization for scramble miRNA (25 nM) in control mouse heart sections. (B) In situ hybridization for U6-snRNA (25 nM) in control mouse heart sections. Scale bar = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57920/57920fig1large.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/57920/57920fig2.jpg" /> 
Figure 2: Cardiomyocyte specific expression of miRNA-182 in control and PlGF mouse hearts. (A-B) In situ hybridization for miRNA-182 in control and PlGF mouse heart sections. (C-D) Immunostaining for cTnT in control and PlGF mouse heart sections that have been previously stained for miRNA-182. (E-F) DAPI counterstaining for nuclear localization in control and PlGF mouse heart sections that have been previously stained for miRNA-182. (G-H) Merged images of miRNA-182 (deep blue), cTnT (red) and DAPI (light blue) staining for the control and PlGF mouse heart sections. Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57920/57920fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization at JoVE.com

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

tissue-specific-mirna-expression-profiling-mouse-heart-sections-using

Nanyang Technological University

Research

JoVE Journal

Genetics

8.1K Views.  University College London. micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Video: Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

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High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay