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, ...

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 ha...

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 ...

wholemount-in-situ-hybridization-for-astyanax-embryos

Research

Education

JoVE Journal

JoVE Core

Biology

Developmental Biology

Evolution

Evolutionary History

SCIENTISTS IN ACTION

6.9K Views.  University of Cincinnati. 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. 

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 ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) and blind cave (cave fish) morphs in this species presents an opportunity to interrogate the mechanisms underlying morphological and behavioral evolution. Cave fish have evolved novel constructive and regressive traits. The constructive changes include increases in taste buds and jaws, lateral line sensory organs, and body fat. The regressive changes include loss or reduction of eyes. melanin pigmentation, schooling behavior, aggression, and sleep. To experimentally interrogate these changes, it is crucial to obtain large numbers of spawned embryos. Since the original A. mexicanus surface fish and cave fish were collected in Texas and Mexico in the 1990s, their descendants have been routinely stimulated to breed and spawn large numbers of embryos bimonthly in the Jeffery laboratory. Although breeding is controlled by food abundance and quality, light-dark cycles, and temperature, we have found that incremental temperature changes play a key role in stimulating maximal spawning. The gradual increase of temperature from 72 &#176;F to 78 &#176;F in the first three days of a breeding week provides two-three consecutive spawning days with maximal numbers of high-quality embryos, which is then followed by a gradual decrease of temperature from 78 &#176;F to 72 &#176;F during the last three days of the spawning week. The procedures shown in this video outline the workflow before and during a laboratory breeding week for incremental temperature stimulated spawning.

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies.

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) ...