Name: wholemount in situ hybridization for astyanax embryos
Uploaded: 2019-03-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Wholemount In Situ Hybridization for Astyanax Embryos at JoVE.com

The authors wish to thank members of the Gross lab for helpful comments on this manuscript. We wish to acknowledge four high school students who utilized this protocol during summer internships in 2017 and 2018, including Christine Cao, Michael Warden, Aki Li, and David Nwankwo. HL was supported by a UC Biology STEM Fellowship during the summer of 2017. This work was supported by grants from the National Science Foundation (DEB-1457630 to JBG), and the National Institutes of Dental and Craniofacial Research (NIH; DE025033 to JBG).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati (Protocol #10-01-21-01).
1. Fixation
<ol>
	<li>Isolate desired number of Astyanax mexicanus embryos from a breeding tank and fix ~50 embryos at a time. If embryos are large and old, it may be necessary to fix 25 at a time to ensure even fixation.</li>
	<li>Depending on the age of the embryo, utilize the IACUC-approved method of anesthesia. For older embryos with a functioning nervous system, sacrifice embryos via anesthetic overdose. Accordingly, place embryos in a solution of ~1% tricaine (buffered to pH 7.4) to minimize pain and discomfort for the organism.</li>
	<li>Once the embryos are unresponsive to touch, replace system water containing tricaine, and add ~1 mL of 1x phosphate-buffered saline (PBS, pH 7.4).</li>
	<li>Remove the PBS solution, and add 1 mL of 4% paraformaldehyde (PFA). Fix embryos overnight at 4 &#176;C. 
	CAUTION: PFA is hazardous (i.e., it is flammable and is a skin and lung irritant), handle with care.</li>
</ol>
2. Dehydration
<ol>
	<li>To dehydrate the embryos, remove the fixative solution and rinse with 1 mL of PBS. Place embryo-containing vials at an angle (between 30&#176; and 45&#176;), on a platform shaker during rinse. Continue to wash the embryos twice, 5 min per wash.</li>
	<li>If embryos still have a chorion, place all 50 embryos into a 100 mm x 25 mm Petri dish, and carefully isolate them from the chorions using two sets of Watchmaker&#8217;s forceps (e.g., #5 forceps) under a microscope. 
	NOTE: During the steps described below, and for the remainder of the protocol, carefully remove all liquid from the previous step using clean, glass Pasteur pipettes before adding the next solution</li>
	<li>Dehydrate the embryos in a series of increasingly concentrated washes of methanol (MeOH) described below. The dilutions are based on a 1 mL total volume with 500 &#181;L of solution going into each 4 mL glass vial. Perform all dehydration steps at room temperature (RT) on platform shaker.
	<ol>
		<li>Carefully remove the PBS solution. Add a 25% MeOH solution (250 &#181;L of MeOH + 750 &#181;L of PBS). Gently shake on a platform shaker for 5 min.</li>
		<li>Carefully remove the 25% MeOH solution. Add a 50% MeOH solution (500 &#181;L of MeOH + 500 &#181;L of PBS). Gently shake on a platform shaker for 5 min.</li>
		<li>Carefully remove the 50% MeOH solution. Add a 75% MeOH solution (750 &#181;L of MeOH + 250 &#181;L of PBS). Gently shake on a platform shaker for 5 minutes.</li>
		<li>Carefully remove the 75% MeOH solution. Add a 100% MeOH solution (1 mL of MeOH). Gently shake on a platform shaker for 5 min. Repeat this step 3 times.</li>
	</ol>
	</li>
	<li>At this point, store dehydrated embryos, as needed, in their glass vials at -20 &#176;C (long term). Alternatively, proceed directly to day 1 of the protocol.</li>
</ol>
3. Day 1: Rehydration
<ol>
	<li>Obtain dehydrated embryos from -20 &#176;C freezer (or proceed directly from step 2.5).</li>
	<li>Sort the embryos using a Pasteur pipette. One can sort based on morphotype (i.e., cave and/or surface), and the number of genes assessed in each experiment. There are usually no more than 12 embryos per vial once sorted. To maintain organization, use colored lab tape to designate vials and pipettes for each gene. Embryos will stay in the same vial throughout the entire protocol. 
	NOTE: Place the tip of the Pasteur pipette in 100% EtOH to sterilize it between uses.</li>
	<li>Set a shaking water bath to 70 &#176;C to be used in a later step. Carefully, draw out the MeOH in vials of sorted embryos and replace with 500 &#181;L of new 100% MeOH. Wash briefly (~1 min) on platform shaker.</li>
	<li>Rehydrate embryos in an increasing concentration of 1x PBS with Tween 20 (PBT, see below) on platform shaker. The dilutions are based on a 1 mL final dilution volume with 500 &#181;L going into each vial.
	<ol>
		<li>Add a 25% PBT solution (250 &#181;L of PBT, 750 &#181;L of MeOH). Gently shake on a platform shaker for 5 min.</li>
		<li>Carefully remove the 25% PBT solution. Add a 50% PBT solution (500 &#181;L of PBT, 500 &#181;L of MeOH). Gently shake on a platform shaker for 5 min.</li>
		<li>Carefully remove the 50% PBT solution. Add a 75% PBT solution (750 &#181;L of PBT, 250 &#181;L of MeOH). Gently shake on a platform shaker for 5 min.</li>
		<li>Carefully remove the 75% PBT solution. Add a 100% PBT solution (1 mL of PBT). Gently shake on a platform shaker for 5 min. Repeat this step 3 times.</li>
	</ol>
	</li>
</ol>
4. Day 1: Digestion and fixation
<ol>
	<li>Prepare a proteinase K (PK) solution by adding 1 &#181;L of PK (20 mg/mL) to 2 mL of PBT.</li>
	<li>In anticipation of subsequent steps, obtain frozen aliquots of hybridization buffers (Hyb- and Hyb+; see Supplemental File 2 and Supplemental File 3) and PFA from -20 &#176;C storage.
	<ol>
		<li>Allow PFA to thaw at RT.</li>
		<li>Place aliquots of Hyb- and Hyb+ in a rotating 70 &#176;C water bath. Place all reagents and vials inside a small &#8220;gasket&#8221; with a mesh bottom, inside the floating water bath apparatus. This enables simple addition and removal of tubes and vials from the rotating 70 &#176;C water bath.</li>
	</ol>
	</li>
	<li>Gently add PK solution to the vial(s) of embryos ensuring all tissues are completely covered with solution. Digest embryos for ~12 min in PK working solution on the platform shaker. 
	NOTE: The length of digestion can be varied by the investigator to ensure optimal results.</li>
	<li>Gently draw off the PK solution, and briefly flood the vial with PBT to dilute any remaining PK.</li>
	<li>Draw off the PBT solution and replace with 500 &#181;L of new PBT. Allow the solution to rinse on the platform shaker for 5 min.</li>
	<li>Draw off PBT and replace with 500 &#181;L of thawed 4% PFA. Allow the embryos to incubate for 20 min on the platform shaker at RT.</li>
	<li>Draw off the 4% PFA, and briefly flood the vial with PBT to dilute any remaining PFA. Draw off the PBT, and replace with 500 &#181;L of fresh PBT. Allow embryos to rinse for 5 min on the platform shaker. Repeat this step 4 more times.</li>
</ol>
5. Day 1: Prehybridization
<ol>
	<li>Place 500 &#181;L of pre-warmed Hyb- solution into the vial. Carefully place the vial in the 70 &#176;C water bath (inside gaskets) without shaking, for 5 min.</li>
	<li>Draw off the Hyb- solution and flood the vial with 500 &#181;L of pre-warmed Hyb+ solution. Place the vial back into the 70 &#176;C water bath with shaking (40 rpm). Incubate for either 4 h, or overnight. 
	NOTE: A 4 h incubation will yield a complete in situ protocol that will last for 4 days in total. Here, this step is presented as an overnight incubation, which will yield a protocol lasting 5 days in total.</li>
</ol>
6. Day 2: Hybridization
<ol>
	<li>Place an aliquot of Hyb+ from the -20 &#176;C freezer into the shaking hot water bath for 5 min.</li>
	<li>Draw off the Hyb+ from the vial and replace with 500 &#181;L of pre-warmed Hyb+. To this solution, carefully add 2 &#181;L of RNA probe to each vial. Gently swirl the vial to ensure even distribution of the probe.</li>
	<li>Incubate the Hyb+ (with added probe) solution in the 70 &#176;C hot water bath overnight while shaking at 40 rpm. 
	NOTE: One can re-use Hyb+ (with probe) solution. For this, take Hyb+ with probe from the first run from the -20 &#176;C freezer and place it in a hot water bath for 5 min. Replace Hyb+ from the day 1 protocol with Hyb+ with probe and allow incubating overnight in hot water bath.</li>
</ol>
7. Day 3: Solution preparation
<ol>
	<li>Prepare microcentrifuge tubes labeled Hyb+ with the &#8220;gene-of-interest&#8221; RNA probe. Prepare the series of dilutions that will be used during day 3.
	<ol>
		<li>Using 6 separate tubes, prepare the following series of dilutions of Hyb- and saline sodium citrate (SSC, 0 to 100%) in a 1 mL volume, and place them in the 70 &#176;C shaking water bath: Tube 1 = 100% Hyb- (1 mL of Hyb-): Tube 2 = 25% 2x SSC (250 &#181;L of 2x SSC, 750 &#181;L of Hyb-); Tube 3 = 50% 2x SSC (500 &#181;L of 2x SSC, 500 &#181;L of Hyb-); Tube 4 = 75% 2x SSC (750 &#181;L of 2x SSC, 250 &#181;L of Hyb-); Tube 5 = 100% 2x SSC (1 mL of 2x SSC); Tube 6 = 100% 0.2x SSC (2 mL of 0.2x SSC). 
		NOTE: Be vigilant of the concentration of SSC, as it changes from 2x to 0.2x.</li>
		<li>Using 4 separate tubes, prepare the following series of dilutions of PBT and SSC in a 1 mL volume, and place at RT: Tube 1 = 25% PBT (250 &#181;L of PBT, 750 &#181;L of 0.2x SSC); Tube 2 = 50% PBT (500 &#181;L of PBT, 500 &#181;L of 0.2x SSC); Tube 3 = 75% PBT (750 &#181;L of PBT, 250 &#181;L of 0.2x SSC); Tube 4 = 100% PBT (1mL of PBT).</li>
		<li>Prepare a tube with 2 mL of maleic acid buffer containing Tween 20 (MABT) working solution.</li>
		<li>Prepare two 15 mL conical tubes of blocking solution. In each tube, add 0.2 g of blocking reagent to 10 mL of MABT (see Supplemental File 4). Place both tubes on a nutating mixer (or platform shaker) until completely dissolved in solution (up to 3 h).</li>
	</ol>
	</li>
</ol>
8. Day 3: Probe removal
<ol>
	<li>Draw off Hyb+ (with probe) solution with a glass Pasteur pipette and place it into a sterile, labeled microcentrifuge tube. Retain this tube in the -20 &#176;C freezer for future use (if probe-labeling is successful).</li>
	<li>Carefully add 500 &#181;L of the warm SSC/Hyb- dilutions (indicated below). Incubate in each of the following solutions for 10 min each in the 70 &#176;C shaking water bath.
	<ol>
		<li>Incubate sequentially with 100% Hyb- (1 mL of Hyb-), 25% 2x SSC (250 &#181;L of 2x SSC, 750 &#181;L of Hyb-), 50% 2x SSC (500 &#181;L of 2x SSC, 500 &#181;L of Hyb-), 75% 2x SSC (750 &#181;L of 2x SSC, 250 &#181;L of Hyb-), 100% 2x SSC (1 mL of 2x SSC), 100% 0.2x SSC (2 mL of 0.2x SSC).</li>
	</ol>
	</li>
	<li>Following the last step, incubate in each of the following solutions for 10 min each. All of the following incubations take place at RT on the platform shaker: 25% PBT (250 &#181;L of PBT, 750 &#181;L of 0.2x SSC), 50% PBT (500 &#181;L of PBT, 500 &#181;L of 0.2x SSC), 75% PBT (750 &#181;L of PBT, 250 &#181;L of 0.2x SSC), 100% PBT (1 mL of PBT).</li>
	<li>After a 10 min incubation, remove the 100% PBT, and add 500 &#181;L of MABT into each vial. Repeat this step twice for 5 min.</li>
</ol>
9. Day 3: Blocking
<ol>
	<li>Remove MABT from each vial and flood with premixed blocking solution from one of the tubes (prepared in step 7.1.4). Place vial on a nutating mixer for ~4 h at RT.</li>
	<li>Add 2 &#181;L of Anti-DIG-AP Fab fragments to the second vial of 10 mL blocking solution (prepared in step 7.1.4) and briefly vortex.</li>
	<li>Fill each vial almost completely with blocking solution (~5 mL) and place on nutating mixer overnight in a refrigerator at 4 &#176;C.</li>
</ol>
10. Day 4: MABT Rinses
<ol>
	<li>Prepare a stock vial of 10% normal goat serum (NGS) in MABT (add 100 &#181;L of NGS to 900 &#181;L of MABT).</li>
	<li>Draw off the blocking solution in each vial and add 500 &#181;L of NGS/MABT mixture into each vial. Allow the embryos to incubate for 25 min at RT on the platform shaker.</li>
	<li>Replace the NGS/MABT mixture with 500 &#181;L of 100% MABT. Incubate for 30 min at RT on the platform shaker. Perform this rinse 11 more times throughout the day every 30 min.</li>
	<li>Fill the vial with 100% MABT, and place on a nutating mixer overnight in a refrigerator or walk-in chamber at 4 &#176;C.</li>
</ol>
11. Day 5: Probe visualization
<ol>
	<li>Prepare a 50 mL aliquot of alkaline phosphatase (AP) buffer (see Supplemental File 5). Combine the following in a 50 mL conical tube wrapped in aluminum foil to limit light exposure: 5 mL of 1 M Tris (pH 9.5), 5 mL of 50 mM MgCl2, 5 mL of 1% Tween 20, 5 mL of 1 M NaCl, 30 mL of ddH2O.</li>
	<li>Remove MABT and replace with 1 mL of AP buffer (tube wrapped in foil). Let it wash for 5 min. Do this twice to ensure complete removal of MABT.</li>
	<li>Remove AP buffer and replace with 1 mL of AP buffer with 3.5 &#956;L 5-bromo-4-chloro-3&#39;-indolyphosphate (BCIP) and 4.5 &#956;L of nitro-blue tetrazolium (NBT). Replace with freshly prepared AP buffer/NBT/BCIP once every hour until reaction is complete. Monitor closely, checking every 15 min, to allow the coloration reaction to take place until the desired level of staining has been achieved. If precipitate begins to form, replace the solution sooner.</li>
	<li>Stop the coloration reaction by rinsing the embryos in fresh 100% AP buffer (without NBT/BCIP) for 5 min. Continue rinses in PBT until optimal levels of signal (with minimal amounts of background staining) are achieved. Continue to rinse specimens with increasing dilutions of PBT in AP Buffer as follows: 25% PBT (250 &#956;L of PBT, 750 &#956;L of AP Buffer), rinse for 5 min; 50% PBT (500 &#956;L of PBT, 500 &#956;L of AP Buffer), rinse for 5 min; 75% PBT (750 &#956;L of PBT, 250 &#956;L of AP buffer), rinse for 5 min.</li>
	<li>Rinse embryos in ~5 mL of 100% PBT on nutating mixer until desired minimum background staining is reached. Switch out with fresh PBT several times. This could take up to several days.</li>
	<li>When rinsing is complete, wash embryos in 500 &#956;L of sterile PBS on a platform shaker. Perform this rinse twice for 5 min. After PBS washes, post-fix the specimens in 500 &#956;L of 4% PFA for 1 h at RT on a platform shaker. Alternatively, fix overnight in 1 mL of 4% PFA in the refrigerator at 4 &#176;C.</li>
	<li>Replace the fixative with fresh, sterile PBS. Perform this rinse at least twice for 5 min. Place the embryos in ~4 mL of 100% sterile PBS, and store long term at 4 &#176;C.</li>
</ol>
12. Imaging
<ol>
	<li>Make up an imaging plate in a Petri dish using 3% agarose and TAE buffer. 
	NOTE: Quantities depend on how many plates are needed. Plates can be reused several times. It is recommended that a shallow rectangular mold is placed in the Petri dish while the gel is cooling in order to create a depression for containing the embryos on the plate.</li>
	<li>Place the embryos on the plate in the PBS. 
	NOTE: It is best to gently pour embryos onto the plate instead of pipetting them out because it has been found that they will stick to the inside of plastic pipettes.</li>
	<li>Use light microscopy in order to visualize each embryo. Use a blunt probe to maneuver embryos to desired position.</li>
	<li>Take an image when the embryo is in desired position. Note that it is important to take images of embryos within a couple weeks of completed staining to avoid potential degradation of stain.</li>
</ol>

<ol>
	<li>Valentino, K. L., Eberwine, J. H., Barchas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization: Application to neurobiology. , (1987).</li><li>Mugrauer, G., Alt, F. W., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+proto-oncogene+expression+during+organogenesis+in+the+developing+mouse+as+revealed+by+in+situ+hybridization.">N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization.</a> The Journal of Cell Biology. 107 (4), 1325-1335 (1988).</li><li>Kerney, R., Gross, J. B., Hanken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+cranial+patterning+in+the+direct&#8208;developing+frog+Eleutherodactylus+coqui+revealed+through+gene+expression.">Early cranial patterning in the direct&#8208;developing frog Eleutherodactylus coqui revealed through gene expression.</a> Evolution &amp; Development. 12 (4), 373-382 (2010).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature Protocols. 3 (1), 59-69 (2008).</li><li>Cheung, M., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+development+is+regulated+by+the+transcription+factor+Sox9.">Neural crest development is regulated by the transcription factor Sox9.</a> Development. 130 (23), 5681-5693 (2003).</li><li>Knight, R. D., Nair, S., Nelson, S. S., Afshar, A., Javidan, Y., Geisler, R., Rauch, G. J., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lockjaw+encodes+a+zebrafish+tfap2a+required+for+early+neural+crest+development.">lockjaw encodes a zebrafish tfap2a required for early neural crest development.</a> Development. 130 (23), 5755-5768 (2003).</li><li>Yang, J. W., Jeong, B. C., Park, J., Koh, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHF20+positively+regulates+osteoblast+differentiation+via+increasing+the+expression+and+activation+of+Runx2+with+enrichment+of+H3K4me3.">PHF20 positively regulates osteoblast differentiation via increasing the expression and activation of Runx2 with enrichment of H3K4me3.</a> Scientific Reports. 7 (1), 8060 (2017).</li><li>Ganju, R. K., Brubaker, S. A., Meyer, J., Dutt, P., Yang, Y., Qin, S., Newman, W., Groopman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#945;-chemokine,+stromal+cell-derived+factor-1&#945;,+binds+to+the+transmembrane+G-protein-coupled+CXCR-4+receptor+and+activates+multiple+signal+transduction+pathways.">The &#945;-chemokine, stromal cell-derived factor-1&#945;, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways.</a> Journal of Biological Chemistry. 273 (36), 23169-23175 (1998).</li><li>Cai, Y., Xin, X., Yamada, T., Muramatsu, Y., Szpirer, C., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignments+of+the+genes+for+rat+pituitary+adenylate+cyclase+activating+polypeptide+(Adcyap1)+and+its+receptor+subtypes+(Adcyap1r1,+Adcyap1r2,+and+Adcyap1r3).">Assignments of the genes for rat pituitary adenylate cyclase activating polypeptide (Adcyap1) and its receptor subtypes (Adcyap1r1, Adcyap1r2, and Adcyap1r3).</a> Cytogenetic and Genome Research. 71 (2), 193-196 (1995).</li><li>Stolt, C. C., Rehberg, S., Ader, M., Lommes, P., Riethmacher, D., Schachner, M., Bartsch, U., Wegner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+differentiation+of+myelin-forming+oligodendrocytes+depends+on+the+transcription+factor+Sox10.">Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10.</a> Genes &amp; Development. 16 (2), 165-170 (2002).</li><li>Kimura, T., Takehana, Y., Naruse, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pnp4a+is+the+causal+gene+of+the+Medaka+Iridophore+mutant+guanineless.">pnp4a is the causal gene of the Medaka Iridophore mutant guanineless.</a> G3: Genes, Genomes, Genetics. 7 (4), 1357-1363 (2017).</li><li>Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+protocol+for+whole-mount+in+situ+hybridization+on+Xenopus+embryos.">A rapid protocol for whole-mount in situ hybridization on Xenopus embryos.</a> Cold Spring Harbor Protocols. (8), pp.pdb-prot4809 (2007).</li><li>Schulz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+Drosophila+testes.">In situ hybridization to Drosophila testes.</a> Cold Spring Harbor Protocols. (8), pp.pdb-prot4764 (2007).</li></ol>

The authors have nothing to disclose.

Owing to the vulnerability of RNA to degradation, one of the most critical steps in the protocol concerns the sterile synthesis of the RNA probe. However, if a probe is carefully generated, and provides good results, it can be reused in subsequent staining reactions. A second crucial step is the careful production of all reagents used throughout the protocol. Since this protocol involves several days and many small steps, it is essential that all reagents are accurately produced, and stored in a sterile manner. Further, it is fundamentally important that the investigator keeps careful track of each step in the protocol. We have found that the provided checklist of steps can be extremely useful in ensuring accurate and precise completion of each aspect of this protocol.
We do not often modify the protocol we presented here. However, investigators may perform probe incubations at different temperatures than the ones suggested (i.e., 70 &#176;C). Slight changes in hybridization temperatures will impact binding of RNA probes, and therefore, seeking the optimal hybridization temperature can positively impact the quality of staining. With respect to troubleshooting, we strongly encourage other investigators to utilize the checklist provided with this article (Supplemental File 1). Maintaining careful records is a necessary first step in ensuring high quality staining. A second minor modification is suggested is to perform the final coloration reaction without rocking (e.g., without placing on a platform shaker or nutator). The reason for this is that periodically we note the production of precipitate, that presumably arises from the AP buffer solution. This precipitate usually overstains to a dark color and creates punctate (non-specific) background on the stained tissue. To minimize the production of this precipitate, we prepare sterilized AP buffer just prior to each reaction. Further, once NBT and BCIP have been added to the buffer, we replace it with fresh buffer and NBT/BCIP every hour until the coloration reaction is completed.
A limitation to the presented method is that a chromatic stain was used for gene expression visualization. We prefer this approach since it is cost-effective, and only requires light microscopy to visualize. If one were interested in evaluating quantitative differences, we suggest they use a fluorescent coloration reaction. This will enable semi-quantitation, for example, through comparison of relative fluorescent units of expression between experiments.
Protocols for in situ hybridization are available widely on the web12,13, as well as in scientific publications. The protocol that we present was developed specifically for our model system, Astyanax mexicanus. We have used this protocol to stain the expression of several dozens of genes, and feel it consistently provides high-quality results. A significant advantage of this protocol is the step-by-step checklist of items to enable the investigator to perform multiple tasks while ensuring accurate completion of each of the steps of this protocol. We hope that this protocol will serve as a helpful resource to other investigators in the field and beyond, and anticipate that this common laboratory technique will support future discoveries linking genotype to phenotype in the blind Mexican cavefish.

In situ hybridization is a common method for staining fixed tissues to visualize gene expression patterns1. This technique has been performed for years in other traditional2 and non-traditional3 model systems, for a variety of biological studies. However, several steps and reagents are necessary to successfully perform this procedure. For investigators who have never performed this technique, initiating the process can be intimidating owing to the many steps involved. Further, the lengthy nature of this procedure lends itself to technical errors, which can be challenging to troubleshoot. 
The overall goal of this article is to present a simple and straightforward method that will render this hybridization technique accessible to a wide audience. To reduce the introduction of errors, we present a straightforward approach that yields high quality gene expression staining and minimizes non-specific background signal. This procedure is similar to other approaches developed in traditional model systems, such as Danio rerio4. Here, we aim to facilitate careful implementation of each step using a downloadable checklist (Supplemental File 1), to promote careful implementation of the protocol. The rationale for doing this is to facilitate organization through the many steps involved in this procedure. This article is appropriate for researchers interested in performing whole-mount in situ hybridization in developing embryos, but have not yet performed the procedure. The advantage of the chosen approach for Astyanax researchers is that it has been tested and proven in both cavefish and surface fish morphs, thereby facilitating comparative expression analyses. The presented method can be used by researchers in studies on Astyanax and other systems.

<table><tbody><tr><td>10 mL Serological Pipette</td><td>VWR</td><td>89130-888</td><td></td></tr><tr><td>1000 mL Filtration Unit</td><td>VWR</td><td>89220-698</td><td></td></tr><tr><td>15 mL Conical</td><td>VWR-Greiner</td><td>82050-278</td><td></td></tr><tr><td>25 mL Serological Pipette</td><td>VWR</td><td>89130-890</td><td></td></tr><tr><td>250 mL Filtration Unit</td><td>VWR</td><td>89220-694</td><td></td></tr><tr><td>5 mL Serological Pipette</td><td>VWR</td><td>89130-886</td><td></td></tr><tr><td>50 mL Conical</td><td>VWR-Falcon</td><td>21008-940</td><td></td></tr><tr><td>500 mL Filtration Unit</td><td>VWR</td><td>89220-696</td><td></td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Sigma-Roche</td><td>11093274910</td><td></td></tr><tr><td>BCIP</td><td>Sigma-Aldrich</td><td>B8503-1G</td><td>1 g</td></tr><tr><td>Blocking Solution</td><td>Sigma-Roche</td><td>11 096 176 001</td><td>50 g</td></tr><tr><td>Citric Acid</td><td>Fisher Scientific</td><td>A104-500</td><td>500 g</td></tr><tr><td>DIG RNA Labeling Kit (SP6/T7)</td><td>Sigma-Roche</td><td>11175025910</td><td></td></tr><tr><td>Eppendorf Tubes</td><td>VWR</td><td>20170-577</td><td></td></tr><tr><td>Ethanol</td><td>Fisher-Decon</td><td>04-355-223</td><td>1 Gal</td></tr><tr><td>Formamide</td><td>Thermo Fisher Scientific</td><td>17899</td><td>100 mL</td></tr><tr><td>Glass dram vials</td><td>VWR</td><td>66011-041</td><td>1 Dr</td></tr><tr><td>Glass Pipettes</td><td>Fisher Scientific</td><td>13-678-8A</td><td></td></tr><tr><td>HCl</td><td>Thermal-ScientificPharmco-AAPER</td><td>284000ACS</td><td>500 mL</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393-25KU</td><td></td></tr><tr><td>Magnesium Chloride-crystalline</td><td>Fisher Scientific</td><td>M33-500</td><td>500 g</td></tr><tr><td>Maleic Acid</td><td>Sigma</td><td>M0375-100g</td><td>100g</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>4L</td></tr><tr><td>Molecular-grade Water (RNase-free)</td><td>VWR</td><td>7732-18-5</td><td>500 mL</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-3</td><td>3 kg</td></tr><tr><td>NaOH pellets</td><td>Fisher Scientific</td><td>S318-500</td><td>500 g</td></tr><tr><td>NBT Substrate powder</td><td>ThermoFisher Scientific</td><td>34035</td><td>1 g</td></tr><tr><td>Normal Goat Serum</td><td>Fisher-Invitrogen</td><td>31873</td><td></td></tr><tr><td>Nutating Mixer</td><td>VWR</td><td>82007-202</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127-500g</td><td>500 g</td></tr><tr><td>PBS 10x</td><td>Fisher Scientific</td><td>BP399-20</td><td>20L</td></tr><tr><td>Proteinase K (200mg/10ml)</td><td>Qiagen</td><td>19133</td><td>10 mL</td></tr><tr><td>Plastic Pipettes</td><td>VWR-Samco</td><td>14670-147</td><td></td></tr><tr><td>RNAse</td><td>Sigma</td><td>R2020-250mL</td><td>250 mL</td></tr><tr><td>Shaking Water Bath 12 L</td><td>VWR</td><td>10128-126</td><td>12 L</td></tr><tr><td>Standard Analog Shaker</td><td>VWR</td><td>89032-092</td><td></td></tr><tr><td>Tris</td><td>Sigma Millipore-OmniPur</td><td>9210-500GM</td><td>500 g</td></tr><tr><td>tRNA Yeast</td><td>Fisher-Invitrogen</td><td>15401011</td><td>25 mg</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416-50mL</td><td>50 mL</td></tr><tr><td>Vortex-Genie 2</td><td>Fisher Scientific-Scientific Industries, Inc</td><td>50-728-002</td><td></td></tr><tr><td>Lithium Chloride (LiCl)</td><td>Sigma-Aldrich</td><td>203637-10G</td><td>10 g</td></tr></tbody></table>

wholemount in situ hybridization for astyanax embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

In this report, we provide a simple and straightforward approach to perform labeling of embryonic Astyanax specimens for high-quality gene expression analysis. This technique can be carried out in either four or five days, and each principal step in the procedure is represented in a color-coded flowchart (Figure 1). Once completed, stained embryos should harbor a dark purple chromatic label in tissues expressing the particular gene of interest. We have successfully implemented this protocol in both Pach&#243;n cavefish (Figure 2A-B,E) and surface fish (Figure 2C-D,F) embryos.
The cavefish embryos were stained for two transcription factors that label early neural crest tissues, Sox9 and Tfap2a5,6. Embryos labeled for Sox9 expression demonstrate clear labeling in the developing branchial arches and pectoral fin (yellow arrowheads, Figure 2A). Note that staining is virtually absent in the yolk sac or the developing somites on the flank (Figure 2A). Similarly, Tfap2a expression is evident in the portions of the developing head, as well as early migration neural crest cells (Figure 2B, arrowhead) along the dorsal flank region of the embryo. The third representative gene presented for cavefish embryos is Phf20a, a marker of osteoblast differentiation7. Note the positive staining in the portions of the somitic mesoderm and posterior head that are destined to give rise to bony tissue (Figure 2E, arrowheads).
In surface fish embryos, we probed for the genes CXCR, Adcyap1a, and Sox10. CXCR encodes a G-protein membrane-bound receptor that binds CXC chemokines8. Positive labeling is present in isolated regions of the head and flank (Figure 2F, arrowheads), as well as a few individual cells overlying the yolk sac. The gene adenylate cyclase-activating polypeptide, Adcyap1a, is expressed in regions of the central nervous system, including pituitary cells. Note the highly specific expression in paired, bilateral clusters of cells on the dorsal aspect of the embryo; as well as a larger region of midline expression (Figure 2C, arrowheads). Finally, we present the expression of Sox10, a transcription factor which labels early neural crest and oligodendrocyte cells10. Highly specific positive staining is evident as an early marker of neural crest on the left and right sides of the dorsal embryo (Figure 2D, arrowheads).
We present each of the two types of confounding issues other investigators may encounter. The first issue one periodically encounters is punctate specks of non-specific labeling. These specks may arise as precipitate from the final MABT rinses, or the AP buffer during the coloration reactions. An example of this non-specific labeling is evident on the yolk sac of a surface fish embryo stained for expression of Pnp4a. This gene encodes an enzyme (purine nucleoside phosphorylase) that facilitates production of iridescent pigmentation11. This gene is first evident in the developing eye and the swim bladder. The punctate specks observed in some surface specimens (Figure 3A), were eliminated by frequent washes and replacement of the AP Buffer + NBT/BCIP in the final stages of the protocol (Figure 3B). A second issue that one periodically encounters is the diffuse, largely non-specific expression of genes that would otherwise produce a distinct expression pattern. One example is the gene BMP4, which appears as a largely diffuse pattern with low levels of chromogen present throughout the specimen (Figure 3C). In cases such as these, we generally identify a different region of the gene, amplify into a vector and perform a new probe synthesis (see Supplemental File 6). The example of a control (no probe) specimen (Figure 3D) is provided to illustrate the diffuse and non-specific nature of our failed BMP4 probe.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59114/59114fig1.jpg" /> 
Figure 1: A simple flowchart for whole-mount in situ hybridization. This flowchart utilizes color-coding to illustrate the principal steps of in situ hybridization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59114/59114fig1large.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/59114/59114fig2.jpg" /> 
Figure 2: Representative staining for six genes, using both cave and surface morphs of Astyanax. (A) Image shows the specific staining (yellow arrows) of Sox9 on the right lateral side of a 72 h post-fertilization (hpf) Pach&#243;n cavefish (45x). (B) Specific staining for Tfap2a is evident on the right lateral side of a 36 hpf Pach&#243;n cavefish (45x). (C)&#160;Bilateral and midline staining (yellow arrowheads) of Adcyap1a are labeled in the dorsal region of a 72 hpf surface fish (100x). (D)&#160;Staining of Sox10 in neural crest tissues of a 24 hpf surface fish (100x). (E)&#160;Phf20a demonstrates a faint, but clear, pattern of expression in the dorsal region of a 24 hpf Pach&#243;n cavefish (100x). (F) The cytokine receptor, CXCR, is expressed in distinct regions of the right lateral side of a 24 hpf surface fish (100x). Scale bars in A, B, E, F = 0.5 mm; scale bars in C, D = 2.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59114/59114fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59114/59114fig3.jpg" /> 
Figure 3: Representative examples of sub-optimal results for whole-mount in situ hybridization. (A) Specific staining is evident alongside non-specific precipitate and/or debris (red arrowhead) on the right lateral aspect of a 72 hpf Pach&#243;n cavefish (100x). (B)&#160;The same probe visualized in A, depicting the same staining patterns without precipitate or background in a 72 hpf Pach&#243;n cavefish (100x). (C)&#160;The right lateral flank of a 72 hpf Pach&#243;n cavefish demonstrates diffuse, non-specific staining for BMP4 at 45x magnification. (D)&#160;A 72 hpf Pach&#243;n cavefish subjected to this protocol, without the addition of probe (45x). Scale bars = 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59114/59114fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Wholemount In Situ Hybridization for Astyanax Embryos at JoVE.com

Wholemount In Situ Hybridization for Astyanax Embryos | Developmental Biology | Video

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

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6.9K Views.  University of Cincinnati. Ons in situ protocol is belangrijk omdat het een eenvoudige manier biedt om genexpressiepatronen te visualiseren met minimale achtergrondkleuring. Deze techniek maakt een ingewikkeld en langdurig protocol relatief eenvoudig uit te voeren. Benchtop set-up suggesties en checklists verstrekt maken dit protocol nog gemakkelijker om correct uit te voeren en te reproduceren.Terwijl de hier getoonde technieken specifiek voor Astyanax mexicanus is, zou het waarschijnlijk aan andere systemen va...

Video: Wholemount In Situ Hybridization for Astyanax Embryos

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Genetics

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Immunology and Infection

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Wholemount In Situ Hybridization for Astyanax Embryos

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