This work was supported by grants from the National Institutes of Health [HD089934, DK108495].

The procedures described throughout this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School.
1. Breeding
NOTE: There are several published methods for breeding15,16,17,20 that could also be used at this step. Prior to breeding, adult fish are maintained on a 10:14 light:dark cycle at 23 &#176;C and fed a pellet diet (see Table of Materials) once daily. Fish can be bred in a recirculating system with mechanical filtration and UV sterilization. Breeding can also be accomplished in static (non-recirculating) tanks, but fish should not be left in a static tank for more than 3 days, as water quality quickly degrades.
<ol>
	<li>Fill a 5 gal tank with fish-ready water (dechlorinated water adjusted to: pH = 7.1 +/- 2, conductivity = 900 +/- 150 &#181;S, temperature = 23 &#176;C).</li>
	<li>Place plastic mesh (see Materials) in the bottom of the tank. If breeding in static tanks, affix a water heater to the side of the tank. If breeding in a recirculating system, place a water heater in the system sump. 
	NOTE: The plastic mesh prevents adults from consuming eggs.</li>
	<li>Place one female and two male A. mexicanus fish of greater than 1 year old into the tank. Allow the fish to acclimate for 30 min.</li>
	<li>Set the temperature of the heater to 24 &#176;C (or 1 &#176;C warmer than the starting temperature).</li>
	<li>After 24 h, increase the temperature by 1 &#176;C.</li>
	<li>After 24 h, increase the temperature by 1 &#176;C. Check the tanks daily for eggs by shining a flashlight into the bottom of the tank. Withhold the fish from food during this 3-day period.</li>
	<li>If spawning has not been induced after 3 days, turn off the heater and allow the water to return to room temperature (RT) before moving the fish to their original tank.</li>
</ol>
2. Hatching Fertilized Eggs
<ol>
	<li>Once eggs are identified in a breeding tank, remove the adults and plastic mesh, and reduce the water to a depth of 10 cm using a beaker or cup. 
	NOTE: To estimate time of spawning, use a transfer pipette to place several eggs into a Petri dish and view them with a stereomicroscope to determine the stage14 and estimate the time of fertilization.</li>
	<li>Move the tank to a convenient work surface and remove any opaque eggs or feces, leaving only the translucent, fertile eggs in the tank. 
	NOTE: The number of fertile eggs can be recorded at this time.</li>
	<li>Fill the tank with fish-ready water (see step 1.1) and add 6-7 drops of methylene blue to the tank as the water fills. 
	NOTE: The final concentration of methylene blue is approximately 1.5 ppm.</li>
	<li>Add a heater and aquarium bubbler (attached to an air pump, with a regulator) to the tank.</li>
	<li>Set the heater to 24 &#176;C and adjust the airstream regulator to produce a gentle stream of bubbles.</li>
	<li>Place a cover on the tank to help maintain the water temperature. 
	NOTE: The eggs should begin hatching within 24 h of the spawning time.</li>
</ol>
3. Transfer of Hatched Larvae to Nursery Containers
<ol>
	<li>Add 20 g of salt (see Materials) to 8 L of fish-ready water (see step 1.1) and stir until dissolved. Fill each 1.5 L nursery container (see Materials) with 1 L of the prepared water.</li>
	<li>Use a transfer pipette to move hatched larvae into prepared nursery containers at a density of 20 fish per container. 
	NOTE: A headlamp can be useful to locate and transfer the hatched larvae.</li>
	<li>After all the visible hatched larvae have been removed, agitate the water in the tank by stirring, and/or blowing water jets into the edges and corners of the tank with the pipette. 
	NOTE: This will help reveal larvae that were missed on the first pass.</li>
	<li>Dispose of unused larvae at this stage using the guidelines of the Office of Laboratory Welfare (OLAW). Add sodium hypochlorite to the tank to achieve a final concentration of 6.15%. Wait at least 5 min before pouring down the sink.</li>
	<li>Label each nursery container with the date and time of fertilization. View nursery containers daily and continue to remove any dead larvae.</li>
</ol>
4. Preparation of Rotifer-based Fish Food
<ol>
	<li>See Table of Materials for information on obtaining rotifers. Follow the referenced protocol to set up, maintain, and harvest rotifers22.</li>
	<li>Prepare fish food by adding 3 mL of algae mixture (see Table of Materials) to 1 L of harvested rotifers. This mixture will be added directly to the nursery containers as a food supply.</li>
</ol>
5. Feeding of Post-larval Fish
<ol>
	<li>When the fish are 5 days post fertilization (dpf), add 3 mL of fish food (prepared in step 4.2) to each nursery container. At the optimal density, rotifers should be visible in dense groups at the corners of the nursery containers, and less apparent at the center of the container. Add additional rotifer mixture until the appropriate density has been reached.</li>
	<li>Check the containers daily for the presence of rotifers and add more if the concentration gets depleted. Continue to remove any dead larvae.</li>
	<li>When fish reach 14 dpf, move them to a tank fit with a recirculating system at a density of 5 fish/L of water. 
	NOTE: The number of surviving post-larval fish can be recorded at this time.</li>
</ol>
6. Whole-mount Immunohistochemistry of Post-larval Fish
<ol>
	<li>Remove post-larval fish of the desired stage from food for 24 h by pouring the nursery container containing the fish through a nylon mesh strainer and placing the strainer into a container with clean fish-ready water (see step 1.1). 
	NOTE: It is essential to remove all of the food. Even small amounts of food in the gut are auto-fluorescent and will influence imaging.</li>
	<li>Collect and euthanize the fish. 
	NOTE: The euthanasia protocol should follow OLAW guidelines and be approved by your Institutional Animal Care and Use Committee. We have noted that tricaine alone does not euthanize post-larval fish, and the fish begin to move when transferred from tricaine to fixative if the fish are not also kept on ice.
	<ol>
		<li>Prepare tricaine solution in a beaker by adding 0.4 g of tricaine-S and 0.8 g of sodium bicarbonate to 1 L of deionized water, and place it on ice.</li>
		<li>Pour the water containing the fish through a nylon mesh strainer to collect the fish. Gently submerge the strainer in ice-cold Tricaine solution and leave it on ice for 10 min.</li>
	</ol>
	</li>
	<li>Fix the euthanized fish.
	<ol>
		<li>Use a transfer pipette with a cut tip to transfer the fish to a conical tube.</li>
		<li>Remove the Tricaine solution with a transfer pipette and replace with fixative. Incubate with rocking. 
		NOTE: Fixative and fixation time must be determined based on the antibody used. 10% formalin solution (4% formaldehyde, see materials) overnight at 4 &#176;C is adequate for the antibodies listed in the Table of Materials. 
		CAUTION: Formalin is toxic and flammable. Wear personal protective equipment (gloves, lab coat, and splash goggles) and handle in a chemical hood. Solutions containing formalin should be disposed as hazardous waste.</li>
		<li>Use a transfer pipette to carefully remove the fixative without disturbing the fish. Add 3 mL of phosphate buffered saline-triton solution [PBS with 0.1% Triton (PBST), see Materials] and incubate for 15 min at RT with rocking.</li>
		<li>Remove PBST and replace with fresh PBST and incubate for 15 min. Repeat this &#8220;washing&#8221; one additional time. 
		NOTE: Fish can be stored in PBS containing 0.02% sodium azide at 4 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Perform whole-mount immunostaining.
	<ol>
		<li>Prepare 50 mL of blocking solution [PB-0.5% Triton X, 0.2% bovine serum albumin (BSA), 1% dimethyl sulfoxide (DMSO), 0.02% sodium azide, 5% donkey serum]. 
		CAUTION: Sodium azide and DMSO are toxic. Personal protective equipment (gloves, lab coat, and splash goggles) should be used when handling. Any solutions should be disposed as hazardous waste.</li>
		<li>Use a transfer pipette to transfer the fish to a 4 mL glass vial with screw-top cap. Use a transfer pipette to remove the PBST and add 3 mL blocking solution. Incubate for 1 h at RT with rocking.</li>
		<li>Use a transfer pipette to remove the blocking solution and add primary antibody diluted in blocking solution. Incubate overnight at room temperature with agitation. 
		NOTE: For example, add 1:250 dilution of anti-HuC/HuD Neuronal Protein Mouse Monoclonal Antibody (see Table of Materials for a list of antibodies that have been successfully used in A. mexicanus). A set of fish with no primary antibody added should be included at this step. Volume of antibody should be enough to cover the fish and allow for agitation.</li>
		<li>Wash the fish 3 times with PBST as described in step 6.3.4. Replace PBST with secondary antibody diluted in blocking solution and incubate overnight at RT with agitation. 
		NOTE: Optimal primary and secondary antibody concentrations, incubation time, and incubation temperature should be determined for each antibody. Overnight at RT was effective for the antibodies listed in Table of Materials.</li>
		<li>Wash the fish 3 times with PBST, with each wash lasting 15 min as described in step 6.3.4.</li>
		<li>Transfer the fish to PBS for short-term storage before proceeding with dissecting, mounting, or sectioning the fish.</li>
	</ol>
	</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+in+cavefish+as+an+adaptation+to+a+nutrient-limited+environment.">Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.</a> Nature. 555 (7698), 647-651 (2018).</li><li>Moran, D., Softley, R., Warrant, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyeless+Mexican+cavefish+save+energy+by+eliminating+the+circadian+rhythm+in+metabolism.">Eyeless Mexican cavefish save energy by eliminating the circadian rhythm in metabolism.</a> PloS One. 9 (9), e107877 (2014).</li><li>Aspiras, A. C., Rohner, N., Martineau, B., Borowsky, R. L., Tabin, C. 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The authors have nothing to disclose.

Comparing gene activity between surface and cave A. mexicanus requires carefully controlled environmental parameters and methods that can be replicated across laboratories. Our protocol for raising A. mexicanus provides consistent nutritional content during post-larval development. Following this feeding regime, gene function can be confidently compared between populations using the robust immunohistochemistry protocol we present. Here we discuss the significance of this method as well as its limitations and future applications.
To achieve density-matched growth, we found that post-larval fish can be raised without recirculating water for two weeks in 1.5 L containers on a diet of rotifers. This protocol can also be used to raise fish on a recirculating system; however, rotifers must be added daily to compensate for those lost through the tank outflow. Newly hatched Artemia nauplii are commonly used as a food source in aquaculture, but we found that using rotifers has considerable advantages, including: reduced price, improved biosecurity, consistent nutrition, and better water quality (see below).
First, the weekly cost in consumables is 4 dollars for rotifers, compared to 14 dollars for Artemia. Regarding biosecurity, rotifers are raised in the laboratory under controlled conditions, while Artemia are collected from the wild and subject to natural variation in microbial or pathogen contents23. Additionally, the nutrient composition of Artemia nauplii are environmentally determined and therefore inconsistent. Nauplii thrive on their own energy stores after hatching; they quickly loose nutritional value as they develop and should optimally be fed to the fish within several hours. Artemia begin feeding at 12 house post-hatching, representing the first time that they could be nutritionally enriched; however, at this stage they have become too large for 5 dpf fish to consume. In comparison, rotifers continuously feed on marine microalgae resulting in high nutrient content regardless of when the rotifers are harvested. Rotifers are much smaller than Artmeia nauplii (160 vs. 400 microns), making them easier for the fish to capture and swallow. Post-larval surface fish and cavefish consume rotifers in similar quantities suggesting no difference in preference or ability to capture the rotifers10.
Finally, Artemia nauplii begin dying in fresh water several hours after being introduced. Uneaten nauplii will decay, rapidly decreasing the water quality if they are not manually removed. Removing dead Artemia is time-consuming and dangerous for post-larval fish that are not much bigger than Artemia and may be accidently removed or injured. Rotifers can live indefinitely in the nursery containers and provide food to the fish at all times without significantly impacting water quality.
While using rotifers as a food source has considerable benefits, to maintain the rotifer stock, algae must be added to the culture system daily. This can be achieved with an automatic feeder that dispenses liquid algae into the rotifer culture container (see Table of Materials). Rotifers must also be harvested from this set-up every 24-48 hours to maintain the health of the culture. Researchers that breed fish very infrequently (once a year, for example) and are not concerned with making comparisons between the populations at post-larval stages may prefer Artemia as a food source, since the encysted embryos can be hatched at any time.
We recommend tracking the number of hatched and surviving larvae to monitor the success of hatching and growth. If most of the embryos or larvae die, it may be due to bacterial or fungal contamination. It is recommended to monitor the water quality of the fish-ready water and sterilize any equipment with 70% ethanol. Nursery containers can be re-used after they are cleaned and sterilized. To minimize risk of disease, it is also critical to remove any dead fish from the nursery containers and not add rotifers before 5 dpf, when the fish begin to eat.
A. mexicanus are sexually mature at approximately one year old. This is a limitation for generating transgenic A. mexicanus compared to Danio rerio (zebrafish) that are able to breed at 10-12 weeks24. Immunohistochemistry (IHC) is an alternative method to investigate gene expression and protein localization. The protocol described here can be adapted at each step and used to detect antigens in any tissue of interest. It is important to note, however, that some tissues may be more difficult to visualize in surface fish due to pigmentation (a barrier that does not exist in unpigmented cavefish), which may influence the interpretation of comparative studies. To address this potential problem, surface fish pigment can be bleached after fixation using 3% hydrogen peroxide.
IHC requires successful tissue fixation, blocking, and antibody penetration. Methods for each vary depending on the tissue and protein of interest. The fixative and fixation time must preserve cell architecture while maintaining the antigen epitope. For this protocol, we use a cross-linking fixative (paraformaldehyde) and omit a denaturing fixative (such as methanol or acetone). We found that incubation in acetone diminished antibody signal for neuronal and pancreatic markers. The blocking step is essential to prevent antibodies from binding to non-target proteins in the tissue. This method uses a combination of normal serum (5%) and BSA (0.2%) in the blocking solution. The blocking solution contains antibodies and proteins that bind to reactive sites on proteins in the tissue, diminishing non-specific binding of the primary and secondary antibodies.
To achieve antibody penetration, the tissue must be permeabilized. This can be achieved with detergents or denaturing solvents but must be optimized to preserve the antigen epitope. Our protocol uses a combination of Triton and dimethyl sulfoxide (DMSO). Triton and DMSO are included during the blocking and antibody incubation steps at concentrations of 0.5% and 1%, respectively. Using this concentration, we have observed staining in the brain, pancreas, intestine, and muscle, suggesting that it is likely effective for penetration of all tissues. Fish size may also influence penetration. This protocol has not been tested on fish that are greater than 14 days old (approximately 7 mm in length). To troubleshoot staining, it is recommended to alter the fixation, blocking, and antibody penetration steps. It is also important to examine the sequence conservation of the immunogen with the A. mexicanus protein of interest using the available genome25.
A. mexicanus is an excellent model to investigate evolution&#160;as populations of the same species that have evolved in dramatically different environments can be directly compared in the laboratory. Standard husbandry protocols, both within and among laboratories, are essential to understanding the biological differences between surface fish and cavefish. Our article provides a method to examine development and gene activity in post-larval fish exposed to consistent growth parameters.

The Mexican tetra, Astyanax mexicanus, is a single species of fish that exists as river-dwelling populations (surface fish) and a number of cave-dwelling populations (cavefish) named for the caves they inhabit (i.e., Tinaja, Molino, Pach&#243;n). A growing number of researchers are using A. mexicanus to investigate the genetic and developmental bases of behavioral1,2,3,4, metabolic5,6,7,8, and morphological evolution9,10,11. Available resources for studies of A. mexicanus include a sequenced and annotated genome12; transcriptome13; developmental staging table14; and methods for breeding15,16,17, creating transgenics18, and editing genes19. Dissemination of additional tools and updated standard protocols will accelerate growth of the cavefish research community (see this methods collection20).
Our goal is to add to the existing repertoire of tools by providing a robust method for assessing gene activity in situ in post-larval A. mexicanus, in a manner comparable between laboratories. There are two challenges to achieving this goal. First, there is a need for standardized regimes for hatching and raising the fish between laboratories, as differences in parameters such as feeding and density affect growth and maturation, thereby impacting gene activity. Second, there is a need for a standardized yet adaptable method for examining patterns of gene activity in the post-larval fish. We address these issues here, establishing standard practices for raising fish to post-larval stages and introducing a robust whole-mount immunohistochemistry (IHC) protocol for assessing gene expression in A. mexicanus.
We first demonstrate how to breed the fish through natural spawning and identify fertilized eggs. Described next is how to hatch fertilized eggs (larvae) and transfer them to nursery containers, where they are maintained at a density of 20 fish per container for two weeks without recirculating or changing the water. At 5-days post fertilization, the fish have developed to post-larval stages (no longer having a yolk supply) and are provided algae-fed Brachionus plicatilis (rotifers) as a nutrient-rich food source that do not require daily replenishment. This method provides consistent growth parameters for larval and post-larval development.
To assess gene function, we demonstrate how to remove the fish from nursery containers and perform whole-mount IHC. The IHC method presented is adapted from protocols developed for use with Danio rerio21 and is effective for examining antigens in all A. mexicanus tissues tested, including the brain, intestine, and pancreas. IHC is a faster alternative to generating transgenic animals for examination of gene expression and protein localization. This protocol will be useful for studies aimed at growing A. mexicanus and comparing the phenotypes of surface fish and cavefish at post-larval stages.

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			<td>Kordon</td>
			<td>B016CBHZUS</td>
			<td>antifungal</td>
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			<td>heater</td>
			<td>Finnex</td>
			<td>4711457836017</td>
			<td>100W Digital Control Heater</td>
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			<td>airstone</td>
			<td>Lee&#39;s Aquarium &#38; Pet Products</td>
			<td>10838125202</td>
			<td>disposable air stone</td>
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			<td>51378014021</td>
			<td>Sea Salt</td>
		</tr>
		<tr>
			<td>nursery container</td>
			<td>IPC</td>
			<td>21545-002</td>
			<td>40 oz or 1.5 L clear containers</td>
		</tr>
		<tr>
			<td>transfer pipette</td>
			<td>VWR</td>
			<td>414004-002</td>
			<td>plastic bulb pipettes</td>
		</tr>
		<tr>
			<td>compact culture system(CCS) starter kit with Brachionus plicatilis (L-type) rotifers</td>
			<td>Reed Mariculture</td>
			<td>na</td>
			<td>fish food</td>
		</tr>
		<tr>
			<td>RGcomplete</td>
			<td>APBreed</td>
			<td>817656016572</td>
			<td>32 oz bottle of rotifer food</td>
		</tr>
		<tr>
			<td>Programmable Auto Dosing Pump DP-4</td>
			<td>Jebao</td>
			<td>DP-4</td>
			<td>automatic feeder for rotifers</td>
		</tr>
		<tr>
			<td>Tricane-S</td>
			<td>Western Chemical</td>
			<td>MS 222</td>
			<td>fish anesthetic</td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761-500G</td>
			<td>for tricane solution</td>
		</tr>
		<tr>
			<td>nylon mesh strainer</td>
			<td>HIC (Harold Import Co.)</td>
			<td>735343476235</td>
			<td>3-inch diameter</td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128-4L</td>
			<td>fixative</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Invitrogen</td>
			<td>AM9625</td>
			<td>buffer, dilute to 1X using distilled water</td>
		</tr>
		<tr>
			<td>Triton-x 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-250ML</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-25G</td>
			<td>anti-bacterial</td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-100G</td>
			<td>blocking reagent</td>
		</tr>
		<tr>
			<td>glass vial with screw-top cap 4mL</td>
			<td>Wheaton</td>
			<td>224742</td>
			<td>staining vial</td>
		</tr>
		<tr>
			<td>plastic mesh screen for breeding tank</td>
			<td>Pentair</td>
			<td>N1670</td>
			<td>Cut into a rectangle 6mm larger on all edges than the dimensions of the bottom of the breeding tank. Cut a 6mm square from each corner of the rectangle. Bend the edges of the screen down along all four edges.Place a pair of 6mm vinyl-coated disk magnets on either side (top and bottom) of the mesh on each corner. The screen should be as snug as possible to the sides of the tank. The screen can be removed from the tank with a metal fish net.</td>
		</tr>
		<tr>
			<td>vinyl-coated disk magnets</td>
			<td>Kjmagnets</td>
			<td>D84PC-AST</td>
			<td></td>
		</tr>
		<tr>
			<td>New Life Spectrum Thera-A pellet fish food</td>
			<td>New Life International</td>
			<td>na</td>
			<td>Adult fish food. A list of retailers for this product is available on the company website</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>insulin antibody from guinea pig</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>glucagon antibody from sheep</td>
			<td>Abcam</td>
			<td>ab36215</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>acetylated tubulin antibody from mouse</td>
			<td>Sigma</td>
			<td>T6793</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>HuD/HuC antibody from mouse</td>
			<td>Life Technologies</td>
			<td>A-21271</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>nitric oxide synthase (nNOS) antibody from rabbit</td>
			<td>Abcam</td>
			<td>ab106417</td>
			<td>5&#956;g/mL</td>
		</tr>
		<tr>
			<td>choline acetyltransferase (ChAT) from rabbit</td>
			<td>Abcam</td>
			<td>ab178850</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>seratonin (5HT) from rabbit</td>
			<td>Immunostar</td>
			<td>20080</td>
			<td>1:500</td>
		</tr>
	</tbody>
</table>

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.

Table 1 shows success during one year of breeding surface fish and Tinaja, Molino, and Pach&#243;n cavefish in static breeding tanks. Surface and Pach&#243;n spawns with fertilized embryos always produced hatched larvae, while Molino and Tinaja were unsuccessful some of the time (2/6 and 2/18 spawning events did not produce hatched larvae, respectively). There is variation in clutch size that does not appear to be attributed to age of the parent fish. Table 2 shows the total number of hatched larvae resulting from some of the spawning events, and the age of the parent fish. In general, we found that surface fish produce the greatest number of larvae per spawn (average 1,550 &#177; 894, n = 5), followed by Pach&#243;n (average 879 &#177; 680, n = 6), Tinaja (average 570 &#177; 373, n = 11), and Molino (average 386 &#177; 276, n =3). The number of larvae produced is typically more than are needed per experiment or for growth into adults. We typically set up 6-18 nursery containers (120-360 larvae) and euthanize the remaining fish.
To measure the success of the nursery protocol we recorded the number of hatched larvae and surviving post-larval fish from successful spawning events. Table 3 shows data from 1 month of breeding in recirculating tanks and includes the number of larvae transferred to nursery containers that survived to 14 dpf. During this month, the survival rate ranged from 41-81 percent, resulting in 65-293 fish available per population for experiments or growth into adults.
To determine if whole-mount immunostaining is successful, we compared the fluorescence of samples incubated with primary antibody to those incubated with secondary antibody only. The fluorescent signal is only visible in the fish incubated with primary antibody. We have used this protocol to successfully label neurons10 (Figure 1) and pancreatic cells6 at stages up to 12.5 dpf in both surface and cave morphotypes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58972/58972fig1.jpg" /> 
Figure 1: Neuron labeling. Whole-mount immunostaining of A. mexicanus. Image of 12.5 dpf surface fish (a) and Pach&#243;n cavefish (b). Image of the mid-body region [hatched yellow outline shown in (a) and (b)] of surface fish (c) and Pach&#243;n cavefish (d) stained with pan-neuronal antibody (Hu). (e) Confocal image of a region of the surface fish intestine showing enteric neurons (Hu) and their projections (acetylated tubulin). For this image, the intestine was dissected out and mounted in medium containing DAPI to stain the nuclei. <a href="https://www.jove.com/files/ftp_upload/58972/58972fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>population</td>
			<td>attempts</td>
			<td>spawning events</td>
			<td>clutches</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>94</td>
			<td>23</td>
			<td>23</td>
		</tr>
		<tr>
			<td>Molino</td>
			<td>110</td>
			<td>6</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>167</td>
			<td>13</td>
			<td>13</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>242</td>
			<td>18</td>
			<td>16</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of data from one year of breeding A. mexicanus in static tanks. Number of breeding attempts, resulting spawning events, and number of spawning events that produced hatched larvae.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>population&#160;</td>
			<td>parent age</td>
			<td>hatched larvae</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>1 year</td>
			<td>989</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>1 year</td>
			<td>1050</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>3 years</td>
			<td>2214</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>3 years</td>
			<td>432</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>3 years</td>
			<td>1768</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>4 years</td>
			<td>2852</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>1 year</td>
			<td>1194</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>1 year</td>
			<td>1933</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>1.5 years</td>
			<td>371</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>3 years</td>
			<td>480</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>4 years</td>
			<td>1190</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>4 years</td>
			<td>110</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>9 months</td>
			<td>259</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>9 months</td>
			<td>253</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>10 months</td>
			<td>1100</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>11 months</td>
			<td>857</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>1 year</td>
			<td>713</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>1 year</td>
			<td>853</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>1 year</td>
			<td>542</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>1.5 years</td>
			<td>360</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>1.5 years</td>
			<td>58</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>1.5 years</td>
			<td>1100</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>4 years</td>
			<td>185</td>
		</tr>
		<tr>
			<td>Molino</td>
			<td>2.5 years</td>
			<td>460</td>
		</tr>
		<tr>
			<td>Molino</td>
			<td>2.5 years</td>
			<td>619</td>
		</tr>
		<tr>
			<td>Molino</td>
			<td>3 years</td>
			<td>81</td>
		</tr>
	</tbody>
</table>
Table 2: Approximate female age and number of hatched larvae from individual spawning events from the indicated populations of A. mexicanus.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>population</td>
			<td>attempts</td>
			<td>spawning events</td>
			<td>clutches</td>
			<td>clutch size</td>
			<td>larvae transferred to nursery cups</td>
			<td>post-larval fish at 14dpf</td>
			<td>survival (%)</td>
		</tr>
		<tr>
			<td>surface</td>
			<td>4</td>
			<td>3</td>
			<td>2</td>
			<td>576 &#38; 1728</td>
			<td>360</td>
			<td>174</td>
			<td>48</td>
		</tr>
		<tr>
			<td>Molino</td>
			<td>4</td>
			<td>2</td>
			<td>1</td>
			<td>228</td>
			<td>159</td>
			<td>65</td>
			<td>41</td>
		</tr>
		<tr>
			<td>Tinaja</td>
			<td>4</td>
			<td>1</td>
			<td>1</td>
			<td>1952</td>
			<td>175</td>
			<td>93</td>
			<td>53</td>
		</tr>
		<tr>
			<td>Pach&#243;n</td>
			<td>4</td>
			<td>1</td>
			<td>1</td>
			<td>1696</td>
			<td>360</td>
			<td>293</td>
			<td>81</td>
		</tr>
	</tbody>
</table>
Table 3: Summary of data from one month of breeding A. mexicanus in a recirculating system and raising the larvae. Number of breeding attempts, resulting spawning events, clutches that produced hatched larvae, average number of larvae per clutch, larvae transferred to the nursery containers, and post-larval fish present in the nursery containers after 14 days.

Watch this Scientific Journal Video about Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry at JoVE.com

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.

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 ...

raising-mexican-tetra-astyanax-mexicanus-for-analysis-post-larval

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JoVE Journal

Genetics

8.6K Views.  Harvard Medical School. 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. 

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

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

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

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

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

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

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

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...