I wish to thank the many undergraduate students who have worked in my laboratory and contributed to the evolution of these protocols. NAT has received support from the Skidmore-Union Network, a project established with a NSF ADVANCE PAID grant.

I. RNA Whole Mount In situ Hybridization in Marine Elasmobranchs
1. Embryo Fixation and Preparation
<ol>
 <li>Skate embryos can be staged according to Ballard 7. Optimal stages for detection of gene expression depends on the tissue of interest. To track gene expression in the skate digestive tract, stages 27-30 are optimal 7.</li>
 <li>Dissect embryos into PBS, separating the embryos from the yolk sac.</li>
 <li>Fix in 30 ml of freshly made 4% paraformaldehyde (PFA) in a 50 ml conical tube at 4 &deg;C with gentle rocking. Fix for 24 hr.</li>
 <li>Wash embryos in PBT, twice for 15 min, rotating at room temperature. All solution recipes for RNA whole mount in situ's are included (Table 1).</li>
 <li>Dehydrate embryos by washing in a methanol series of 25%, 50% and 75% methanol:PBT rocking at room temperature. The duration of the washes depends on the stage of the embryos. For pre-neurulation staged embryos, 5 min of each wash in the methanol series is sufficient. For embryos older than stage 30, washes can last up to 30 min. To determine if embryos are sufficiently penetrated and acclimated to each wash, hold the tube upright in your hand. If the embryos sink to the bottom of the tube, then you are ready for the next wash. If the embryos float to the top of the solution, change the solution and repeat that particular dehydration step.</li>
 <li>Wash twice in 100% methanol for 10 min gently rocking.</li>
 <li>Once dehydrated to 100% methanol, embryos should be stored at -20 &deg;C for at least 24 hr before proceeding with the protocol. Embryos have been successfully stored at -20 &deg;C and used for RNA whole mounts for up to 1 year.</li>
</ol>
2. Synthesis of RNA Probe
<ol>
 <li>Generate a linear fragment of the gene whose expression you want to detect. This can be done either by PCR amplification or linearizing a plasmid with your gene of interest. To amplify by PCR, choose primers whose binding sites flank the gene insert and retain RNA polymerase binding sites in the amplified region. For commonly used plasmids such as pBS or pGEM, M15 forward and reverse primers are ideal. Alternatively, linearize a plasmid by digesting 15 &mu;l of QIAGEN miniprep DNA with an enzyme that cleaves at the 5' end of the gene. Gel extract and purify using the appropriate QIAquick kit.</li>
 <li>Reverse transcribe the RNA probe using 8 &mu;l of linear plasmid or 100 ng of PCR product as a template using the appropriate RNA polymerase (see Table 2 for transcription reaction).</li>
 <li>Incubate reverse transcription reaction for 2 hr at 37 &deg;C.</li>
 <li>To confirm reaction, run 1 &mu;l of the transcription reaction on a 1% agarose/TAE gel. Run gel at 100 mVolts until the dye has just run into the gel. A single prominent band should be visible. The linear DNA template fragment may be faintly visible at a higher molecular weight.</li>
 <li>Add 1 &mu;l of RNAse-free DNAse I to the transcription reaction and incubate at 37 &deg;C for 15 min.</li>
 <li>To precipitate, add 100 &mu;l TE pH 8, 10 &mu;l 4 M LiCl, 300 &mu;l 100% EtOH and incubate at -20 &deg;C for at least 60 min or overnight.</li>
 <li>Spin for 10 min in a 4 &deg;C microfuge at 14,000 rpm.</li>
 <li>Wash pellet with 70% EtOH and air dry.</li>
 <li>Resuspend RNA probe in 20 &mu;l of dH2O. Add 80 &mu;l of hybridization buffer (hybridization buffer should be pre-warmed to 70 &deg;C). The probe can be stored in 80% hybridization buffer for several months at -20 &deg;C or longer at -80 &deg;C.</li>
</ol>
3. Embryo Pre-treatment and Hybridization
<ol>
 <li>Remove embryos stored in 100% methanol from -20 &deg;C. For younger embryos proceed with step (3.2). For later staged embryos (stage 29/30 &lt;) you may see an epidermis layer that has 'lifted' off the body of the embryo. Under the microscope, carefully dissect off the epidermal layer to maximize probe penetration.</li>
 <li>Place embryos in clean glass scintillation vials. Rehydrate the embryos in the reverse methanol series describe above: (75%, 50%, 25% methanol:PBT) by rocking gently each solution for 5-30 min each at room temperature. Complete rehydration with two washes of PBT for 10 min each, gently rocking.</li>
 <li>To remove any pigment, bleach embryos with 6% hydrogen peroxide in PBT for 1 hr at room temperature, rocking.</li>
 <li>Remove hydrogen peroxide by washing three times in PBT, (rocking at room temperature, 10 min each).</li>
 <li>Treat embryos with proteinase K to allow for penetration of probe during hybridization. The amount of proteinase K and the duration of the treatment depends on the tissue you want to examine and the age of the embryo. More superficial tissue and younger embryos require shorter and less proteinase K, while deeper tissues and older embryos require a higher concentration and longer treatment time to fully permeabilize the embryo for probe hybridization. For example, to detect Shh in the gut endoderm of stage 29 skate embryos, 30 &mu;g/ml of proteinase K/PBT is incubated for 20 min at room temperature.</li>
 <li>Quickly rinse embryos in PBT to wash away proteinase K.</li>
 <li>To deactivate proteinase K, postfix embryos with 4% PFA, 0.2% glutaraldehyde in PBT for 20 min, gently rocking.</li>
 <li>Wash twice for 10 min with PBT.</li>
 <li>
 <ol class="lalpha">
 <li>For pre-hybridization, add 2-3 ml of pre-heated hybridization solution to the embryos, just enough to cover them. Rock gently in a heated water bath at 70 &deg;C for 1 hr.</li>
 <li>To prepare hybridization solution, preheat RNA probe to 70 &deg;C for 10 min and cool on ice. Dilute 15-20 &mu;l of RNA probe to 2 ml of fresh preheated hybridization solution, to obtain a final concentration of 0.5-1.0 &mu;g/ml. Remove pre-hybe and add diluted RNA probe to embryos. Embryos should be just covered by solution, add more hybridization solution if necessary. Tightly secure lid of scintillation vial. Hybridize by rocking gently in a heated water bath at 70 &deg;C overnight.</li>
 </ol>
 </li>
</ol>
4. Post Hybridization Washes and Antibody Hybridization
<ol>
 <li>Remove hybridization solution with probe from embryos and place in a fresh scintillation vial or Eppendorf tube. The probe can be stored at -20 &deg;C and reused in the future. For post-hybridization washes, place embryos in a Netwell seated in a 6-well tissue culture plate.</li>
 <li>Wash 3 times for 30 min each in pre-warmed Solution #1, rocking at 70 &deg;C.</li>
 <li>Wash 3 times for 30 min each in pre-warmed Solution #2, rocking at 70 &deg;C.</li>
 <li>Wash 3 times for 5 min each in TBST, rocking at room temperature.</li>
 <li>Pre-block with 10% heat-inactivated sheep serum in TBST for 1 hr, rocking at room temperature.</li>
 <li>Transfer embryos from Netwell to a fresh glass scintillation vial. Add anti-DIG Fab Fragments (1:5,000) in 1% heat-inactivated sheep serum/TBST to embryos. Rock overnight at 4 &deg;C.</li>
</ol>
5. Post Antibody Hybridization Washes
<ol>
 <li>Remove antibody and discard. Place embryos back into Netwell for washes.</li>
 <li>Wash 3 times for 5 min each in TBST, rocking at room temperature</li>
 <li>Wash at least 6 times for 1 hr each, rocking at room temperature.</li>
 <li>Wash overnight in TBST, rocking at 4 &deg;C. As the post antibody washes help cut down on background staining, maximizing the number and duration of washes is preferable. Embryos are routinely washed in TBST at 4 &deg;C, over the weekend.</li>
</ol>
6. Detection of Probe
<ol>
 <li>Make up fresh NTMT solution and filter sterilize.</li>
 <li>Discard TBST wash solution and place embryos in a clean glass scintillation vial. Wash embryos 3 times in fresh NTMT for 10 min each at room temperature, rocking.</li>
 <li>Remove NTMT wash and add reaction mix of 1 x NBT / 1 x BCIP in NTMT. As these reactants are light sensitive, cover scintillation vial in foil and rock at room temperature.</li>
 <li>Monitor color reaction every 15 min until desired gene expression is clearly visible. Strong probes may take as little as 10 min to develop. Weak probes may take 1-2 hr. Do not let reaction go for too long or background staining will begin to appear (the whole embryo starts to turn a dull purple or brown color).</li>
 <li>Wash 2 times for 10 min each in PBT at room temperature, rocking.</li>
 <li>Postfix embryos in 4% paraformaldehyde, 0.1% glutaraldehyde for 1 hr at room temperature. Embryos can be store indefinitely in fixative at 4 &deg;C.</li>
 <li>Visualize embryos with a dissecting stereomicroscope. To produce a light blue background for pictures, place embryos in a dish with pre-hardened 1% agarose/PBS.</li>
</ol>
7. Representative Results for I. RNA Whole Mount in situ hybridization in marine elasmobranchs
RNA whole mount in situ's depicting expression of Sonic hedgehog (Shh) and Hoxa13 in skate embryos are shown in Figure 1. Expression of Shh in higher vertebrates is found in the notochord and gut endoderm and this expression pattern is conserved in the skate (Figure 1a) 8,9 . Marine elasmobranchs have a unique method of osmoregulation that uses the rectal gland to secrete salts. Hoxa13 expression is high in the developing rectal gland (Figure 1b) 10. Hoxa13 gene product role in patterning the rectal gland remains unknown.
II. Paraffin Embedding and Sectioning Elasmobranch Tissue
1. Harvest and Preparation of Tissue
<ol>
 <li>Dissect desired tissue and fix in 10% formalin for 24-48 hr, rocking at room temperature. 4% paraformaldehyde (PFA) can also be used as a fixative (see Discussion).</li>
 <li>Wash out fixative by washing in PBS, 3 times for 30 min each. Depending on the size of the tissue, washing times can be decreased to 10 min or increased to 1 hr.</li>
 <li>Dehydrate embryos by washing in an ethanol series of 25%, 50% and 75% ethanol:PBS rocking at room temperature. Again, the time for each wash will depend on the size of the tissue. For 0.5 cm3 tissue, incubate each wash for 15 min. Increase time for larger pieces. If long term storage of tissue is desired, wash an additional two times in 75% ethanol and store in -20 &deg;C for up to one year. Otherwise proceed to next step.</li>
 <li>Further dehydrate by washing 3 times in 100% ethanol for 15 min each, rocking.</li>
</ol>
2. Paraffin Embedding and Sectioning
<ol>
 <li>Clarify tissue by washing 2 times in xylene for 5 min each in glass scintillation vials with screw cap lids. Xylene will render the tissue brittle, so do not exceed a total of 10 min for washes. The tissue should look translucent when you hold it up to a light. If the tissue is still very opaque after the two washes, do an additional wash in xylene for 5 min and then proceed with protocol. Only handle xylene inside a fume hood with gloves.</li>
 <li>Remove xylene and fill the scintillation vial 2/3 full with melted paraffin. Incubate in a 60 &deg;C oven for 30 min. When putting paraffin into the vial, you will notice the paraffin solidify along the sides of the vial. Place the vial in the oven and allow the paraffin to re-dissolve for a few minutes. Take the vial out of the oven and give the solution a quick swirl to mix and replace in oven for remainder of incubation.</li>
 <li>Repeat paraffin incubations two more times for 30 min.</li>
 <li>Incubate in paraffin twice for an hour each. Change paraffin a final time and incubate overnight in the 60 &deg;C oven.</li>
 <li>The following day, place the tissue in a heated paraffin bath chamber and place under vacuum for 2-3 hr. This will facilitate infiltration of paraffin into the tissue and remove any air bubbles. For some types of thin tissue, a vacuum step may not be required, but it is necessary for whole embryos or digestive tract and other organs with luminal compartments.</li>
 <li>After vacuum-facilitated infiltration of tissue, place tissue in a metal mold with melted paraffin, and cool on an ice plate. Leave on ice or at 4 &deg;C overnight before trying to remove the tissue from the mold. Paraffin blocks of tissue can be stored indefinitely at 4 &deg;C.</li>
 <li>Place paraffin block onto a microtome and cut 8 &mu;m sections. Sections should be floated on a water bath heated to 48 &deg;C and mounted onto glass Superfrost*Plus slides.</li>
 <li>Dry slides overnight in a 37 &deg;C oven.</li>
 <li>Store sections at 4 &deg;C until needed.</li>
</ol>
III. Alcian Blue / Nuclear Fast Red Stain of Elasmobranch Tissue
1. Alcian Blue Stain for Mucins
<ol>
 <li>Place slides with sections facing upward on a slide warmer. Melt for 45 min.</li>
 <li>Place slides into a slide holder. All consequent solutions are contained in tubs inside a fume hood. Slides are washed by transferring them from one tub with a solution to another. Make sure that the solution levels in the tubs covers the slides; the tissues on the slides are completely submerged. Dissolve paraffin by washing slides 2 times in xylene for 5 min each.</li>
 <li>Wash slides two times with 100% ethanol for 1 min each.</li>
 <li>Rehydrate slides in an ethanol series (75%, 50%, 25% ethanol) washing in each solution for 1 min. Wash in dH2O for 1 min.</li>
 <li>Stain in Alcian Blue Solution, pH 2.5 for 15 min.</li>
 <li>Develop stain by washing in running tap water for 3 min. Dip once in dH2O.</li>
 <li>Counterstain in Nuclear Fast Red Counterstain solution for 10 min.</li>
 <li>Wash in running tap water for 3 min and dip in dH2O one time.</li>
 <li>Dehydrate in an ethanol series (25%, 50%, 75%, 100%, 100%) for 20 dips each, or 1 min each. Wash 2 times in xylene for 5 min each. Mount with DPX mounting medium and coverslips. Allow mounting medium to dry and harden for 48 hr in the fume hood before manipulating slides.</li>
</ol>

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I have nothing to disclose.

The protocols presented are classic methods for monitoring gene expression and identifying differentiated cell types, and have been adapted for use in marine elasmobranchs. Further modifications of these protocols may be needed to adapt to different elasmobranch species.
The most common concern regarding RNA whole mount in situ's is the risk of RNase contamination and thereby the degradation of the RNA probe and endogenous messages. Two aspects need to be considered: the synthesis of the riboprobe and its hybridization to embryonic messages, and general techniques for handling RNA. In general, contamination by ribonucleases can be avoided by using plasticware from unopened containers, and previously unused glassware. If new glassware is unavailable, treat all glassware with RNase-AWAY (VWR#17810-491). Washes are done by gently pouring off solutions through a perforated spoon and adding fresh solution to the vial. Alternatively, very small embryos or organs/tissues can be placed in a Netwell that fits into a 6-well tissue culture dish. The tissue can be moved from one solution to the next by simply lifting the Netwell from one well to the next. This helps prevent the loss of any tissue by pouring, and also preserves the architecture of the tissue. Additional suggestions for handling RNA and preventing RNase contamination can be found in Molecular Cloning; A Laboratory Manual, and on several pharmaceutical websites, including Roche (<a href="http://www.roche-applied-science.com/labfaqs/p2_1.htm" target="_blank">http://www.roche-applied-science.com/labfaqs/p2_1.htm</a>) 11.
Generation of the RNA probe and pre-treatment and hybridization of embryos are the most vulnerable steps to contamination by ribonucleases. Antisense RNA probes that complement native mRNA transcript are synthesized by in vitro reverse transcription in the presence of digoxigenin-UTP. Briefly, cDNA plasmids are linearized with a unique restriction enzyme whose site is located at the 5' end of the gene. This allows for the RNA polymerase to fall off at the end of the gene. Choose a RNA polymerase by identifying the RNA polymerase promoter available in the plasmid, just 3' to the stop codon. While T3 and T7 RNA polymerases are often used for pBluescript, SP6 is the choice for genes subcloned into the directional pGEM plasmids. In addition to RNase inhibitor, DEPC-treated water is used in the transcription reaction and purification of the probe. Hybridization solution and steps using PBT in the pre-treatment of embryos should all be made with DEPC water. To DEPC treat water, add 1 ml of DEPC to 1 L of water in a large flask and mix well. Leave overnight in a hood and autoclave the next day. Solutions such as 10 x PBS and SSC may also be made with DEPC-treated water. After hybridization with the riboprobe, the use of RNase-free solutions and similar precautions are no longer necessary.
Additional variables worth considering are the concentration and length of proteinase K treatment, which can affect the penetration of the probe. A careful titration of the proteinase K stock is recommended to optimize treatment time for different embryonic stages. The protocol described here has been routinely and successfully used with embryos at stages 20 - 31 according to Ballard 7. For embryos that are very young or for tissue that is particularly "sticky", the detergent Tween-20 can be replaced with Triton-X. In addition, gene-specific modifications to the protocol may be needed depending on the abundance of transcript. To reduce background, 10% dimethylformamide has been routinely added to the development solution (NTMT) by several groups 12.
In contrast to the sensitive nature of RNA in situ's, the joy of histology is that it is quick and pretty foolproof! Any variability in staining is likely due to inadequate fixation. To ensure that tissue is fully fixed, it is recommended that very large tissues (such as the digestive tract of an adult animal) be dissected into smaller pieces and fresh fixative solution replaced every 10 hr over a 48 hr period. It may be necessary to optimize conditions for fixation, both under and over-fixed tissues can result in poor sectioning or uneven staining. In addition, different histochemical stains work optimally with different fixatives. Thus, it is worth modifying the fixative according to the histochemical stain used 13. 4% paraformaldehyde is recommended when fixing soft tissue organs or young embryos. For older embryos or whole animals, 10% formalin is preferred. Once tissue is paraffin embedded and sectioned, it may be used for multiple purposes, including RNA in situ's. This allows for resolution of gene expression on the cellular level. Alternatively, sections may be stained with different histochemical stains to identify differentiated cell types.
The alcian blue and nuclear fast red staining has been successfully performed in the skate (Leucoraja erinacea), dogfish (Squalas acanthias), hagfish (Myxine glutinosa) and lamprey (Petromyzon marinus), (unpublished results) 10,14 . In addition to the alcian blue pH 2.5 stain described here, modifying the pH of the alcian blue solution can distinguish different mucosaccharides (i.e. sialo- versus sulfomucins) 13. Different mucosaccharide constituents can distinguish goblet cells by function and localization within the digestive tract 15,16 . Alcian blue stain in the digestive tract has been used to diagnose conditions such as Barrett's esophagus and to enhance staining of pancreatic beta-cell granules 17,18 .
In summary, histochemistry and RNA whole mount in situ's when used together can lead to a better understanding of the link between where a gene is expressed during development and the resulting differentiated cell type. Expression of posterior Hox genes has been linked to specification of acid mucin goblet cells in the development digestive tract 10,19 . Here I provide an example of the expression of Shh and a Hox gene along with the presence of acid mucin-producing goblet cells in the L. erinacea digestive tract. These techniques can be widely applied to different species of elasmobranchs, different developmental patterning genes and different histological stains. The continued application and adaptation of techniques to marine elasmobranchs is important to keep pace with the broad use of these animals for biomedical research models in physiology, endocrinology, toxicology and genomics.

<table><tbody><tr><td>10 x transcription buffer</td><td>Roche</td><td>11-465-384-001</td><td></td></tr><tr><td>DIG-RNA labeling mix</td><td>Roche</td><td>11-277-073-910</td><td></td></tr><tr><td>RNAse inhibitor</td><td>Roche</td><td>03-335-399-001</td><td></td></tr><tr><td>RNA polymerase - SP6</td><td>Roche</td><td>10-810-274-001</td><td></td></tr><tr><td>DNAseI, RNAse-free </td><td>Roche</td><td>10-776-785-001</td><td></td></tr><tr><td>Yeast RNA</td><td>Invitrogen</td><td>15401-029</td><td></td></tr><tr><td>CHAPS</td><td>EMD-Millipore</td><td>220201</td><td></td></tr><tr><td>heparin</td><td>Sigma-Aldrich</td><td>H4784</td><td></td></tr><tr><td>DEPC (diethyl pyrocarbonate)</td><td>Research Organics</td><td>2106D</td><td></td></tr><tr><td>Moria Perforated Spoon</td><td>Fine Science Tools</td><td>10370-17</td><td></td></tr><tr><td>Netwell inserts</td><td>Electron Microscopy Sciences</td><td>64713-00</td><td>Netwells for use in 6-well tissue culture dishes</td></tr><tr><td>6-well tissue culture plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Glass scintillation vials with screw-cap lids</td><td>Weaton Science Products</td><td>986540</td><td></td></tr><tr><td>formamide</td><td>Fisher</td><td>BP227500</td><td></td></tr><tr><td>Proteinase K</td><td>Invitrogen</td><td>59895 (AM2542)</td><td></td></tr><tr><td>NBT</td><td></td><td>11585029001</td><td></td></tr><tr><td>BCIP</td><td>Roche</td><td>11585002001</td><td></td></tr><tr><td>Hydrogen peroxide, 30%</td><td>EMD</td><td>HX0635-1</td><td></td></tr><tr><td>Sheep serum</td><td>VWR</td><td>101301-478</td><td></td></tr><tr><td>glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>tRNA</td><td>Roche</td><td>10-109-541-001</td><td></td></tr><tr><td>Anti-DIG Fab Fragments</td><td>Roche</td><td>1137-6623</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 3. Reagents and equipment for RNA whole mount &lt;em&gt;in situ&lt;/em&gt;&#039;s&lt;/strong&gt;.</td></tr><tr><td>1% Alcian Blue 8GS, pH 2.5</td><td>Electron Microscopy Sciences</td><td>26323-01</td><td></td></tr><tr><td>Nuclear Fast Red</td><td>Electron Microscopy Sciences</td><td>26078-05</td><td></td></tr><tr><td>DPX Mountant</td><td>Electron Microscopy Sciences</td><td>13510</td><td></td></tr><tr><td>Paraffin (Paraplast X-tra)</td><td>McCormick Scientific</td><td>39503002</td><td></td></tr><tr><td>10% Formalin, NBF</td><td>VWR</td><td>95042-908</td><td></td></tr><tr><td>Glass scintillation vials with screw-cap lids</td><td>Weaton Science Products</td><td>986540</td><td></td></tr><tr><td>Stainless steal base molds</td><td>Tissue-Tek</td><td>4161-4165</td><td>Multiple sizes available.</td></tr><tr><td>Cassettes </td><td>Tissue-Tek</td><td>4170</td><td></td></tr><tr><td>Slide warmer</td><td>Fisher-Scientific</td><td>12-594Q</td><td></td></tr><tr><td>Tissue Embedder</td><td>Leica Microsystems</td><td>EG1160</td><td></td></tr><tr><td>Microtome, rotary</td><td>Leica Microsystems</td><td>RM2235</td><td></td></tr><tr><td>Tissue-Tek Slide Staining Set</td><td>Electron Microscopy Sciences</td><td>62540-01</td><td></td></tr><tr><td>Tissue-Tek 24-Slide Holder</td><td>Electron Microscopy Sciences</td><td>62543-06</td><td></td></tr><tr><td>Superfrost*Plus slides</td><td>Fisherbrand</td><td>12-550-17</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 4. Reagents and equipment for Alcian Blue stain&lt;/strong&gt;.</td></tr></tbody></table>

rna in situ hybridization in whole mount embryos and cell histology adapted for marine elasmobranchs

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

Examples of alcian blue staining in different regions of the L. erinacea digestive tract are shown in Figure 2. Acid mucin containing globlet cells are clearly visible by the alcian blue stain throughout the digestive tract. The distribution of acid mucins differs in the different regions of the digestive tract, thus reflecting differences in function. Acidic mucins are sparsely produced in the spiral intestine and cloaca, while a high concentration of acid mucins are detected in the distal intestine (compare Figure 2a and 2c with 2b).
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td fo:width="1in">PBT</td>
 <td fo:width="4in">1 x PBS</td>
 </tr>
 <tr>
 <td></td>
 <td>0.1% Tween-20</td>
 </tr>
 <tr>
 <td></td>
 <td>Filter-sterilize in Nalgene-type tissue culture filter units.</td>
 </tr>
 <tr>
 <td>20 x SSC, pH 4.5</td>
 <td>3 M NaCl</td>
 </tr>
 <tr>
 <td></td>
 <td>0.3 M NaCitrate</td>
 </tr>
 <tr>
 <td></td>
 <td>Adjust pH with citric acid.</td>
 </tr>
 <tr>
 <td>Hybridization solution</td>
 <td>50% formamide</td>
 </tr>
 <tr>
 <td></td>
 <td>1.3 x SSC, pH 4.5</td>
 </tr>
 <tr>
 <td></td>
 <td>5 mM EDTA, pH 8</td>
 </tr>
 <tr>
 <td></td>
 <td>50 mg/ml tRNA</td>
 </tr>
 <tr>
 <td></td>
 <td>0.2% Tween-20</td>
 </tr>
 <tr>
 <td></td>
 <td>0.5% Chaps</td>
 </tr>
 <tr>
 <td></td>
 <td>100 mg/ml heparin</td>
 </tr>
 <tr>
 <td></td>
 <td>Aliquot and store at -20 &deg;C for up to one year.</td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>10 mg/ml stock solution</td>
 </tr>
 <tr>
 <td></td>
 <td>20 &mu;l aliquots into 0.5 &mu;l Eppendorf-like tubes and store at -20 &deg;C.</td>
 </tr>
 <tr>
 <td>Solution #1</td>
 <td>50% formamide</td>
 </tr>
 <tr>
 <td></td>
 <td>1.3 x SSC, pH 4.5</td>
 </tr>
 <tr>
 <td></td>
 <td>0.2% Tween-20</td>
 </tr>
 <tr>
 <td></td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Solution #2</td>
 <td>50% formamide</td>
 </tr>
 <tr>
 <td></td>
 <td>1 x SSC, pH 4.5</td>
 </tr>
 <tr>
 <td></td>
 <td>0.2% Tween-20</td>
 </tr>
 <tr>
 <td></td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Sheep serum</td>
 <td>Inactivate by heating to 55 &deg;C for 1 hr, and store in aliquots at -20 &deg;C.</td>
 </tr>
 <tr>
 <td>10 x TBS</td>
 <td>80 g NaCl</td>
 </tr>
 <tr>
 <td></td>
 <td>2 g KCl</td>
 </tr>
 <tr>
 <td></td>
 <td>250 ml, 1 M Tris-HCl, pH 7.5</td>
 </tr>
 <tr>
 <td></td>
 <td>Add water to 1 L</td>
 </tr>
 <tr>
 <td>1 x TBST</td>
 <td>1 x TBS</td>
 </tr>
 <tr>
 <td></td>
 <td>0.1% Tween-20</td>
 </tr>
 <tr>
 <td>NTMT</td>
 <td>100 mM NaCl</td>
 </tr>
 <tr>
 <td></td>
 <td>100 mM Tris-HCl, pH 9.5</td>
 </tr>
 <tr>
 <td></td>
 <td>50 mM MgCl2</td>
 </tr>
 <tr>
 <td></td>
 <td>0.1% Tween-20</td>
 </tr>
 <tr>
 <td></td>
 <td>Make approximately 100 ml fresh before using and filter.</td>
 </tr>
 <tr>
 <td>200 x NBT stock</td>
 <td>50 mg/ml NBT in 70% dimethyl formamide</td>
 </tr>
 <tr>
 <td></td>
 <td>Store in -20 &deg;C in 1 ml aliquots.</td>
 </tr>
 <tr>
 <td>200 x BCIP stock</td>
 <td>25 mg/ml BCIP in water</td>
 </tr>
 <tr>
 <td></td>
 <td>Store in -20 &deg;C in 1 ml aliquots.</td>
 </tr>
 </tbody>
</table>
Table 1. RNA whole mount in situ solutions.
<table border="1">
 <tbody>
 <tr>
 <td>Linearized plasmid</td>
 <td>8 &mu;l</td>
 </tr>
 <tr>
 <td>10 x transcription buffer</td>
 <td>4 &mu;l</td>
 </tr>
 <tr>
 <td>DIG-UTP nucleotide mix</td>
 <td>2 &mu;l</td>
 </tr>
 <tr>
 <td>RNAse inhibitor</td>
 <td>0.5 &mu;l</td>
 </tr>
 <tr>
 <td>RNA polymerase (SP6, T3 or T7)</td>
 <td>1 &mu;l</td>
 </tr>
 <tr>
 <td>RNAse-free sterile H20</td>
 <td>4.5 &mu;l</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>20 &mu;l</td>
 </tr>
 </tbody>
</table>
Table 2. Transcription reaction to generate RNA probe.
<img src="/files/ftp_upload/50165/50165fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50165/50165fig1highres.jpg"/> 
 Figure 1. Shh and Hoxa13 expression in Leucoraja erinacea embryos are visualized by RNA whole mount in situ hybridization. (a) Shh expression is detected in a stage 29 skate embryo. (a') Higher magnification of (a) reveals Shh expression in the notochord (arrowheads) and gut endoderm (arrows). (b) Hoxa13 expression is localized to the rectal gland (arrow) of a stage 29 skate embryo. (b') Dissected digestive tract from the embryo in (b) demonstrates the specificity of Hoxa13 transcript expression to the rectal gland. e, endoderm; n, notochord; rg, rectal gland. <a href="https://www.jove.com/files/ftp_upload/50165/50165fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50165/50165fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50165/50165fig2highres.jpg"/> 
 Figure 2. Distribution of acidic mucin-producing goblet cells in the skate digestive tract. (a, c) Low numbers of acidic mucin-producing goblet cells are detected by the alcian blue stain in the spiral intestine and cloaca, respectively (arrows). (b) The distal intestine contains a higher density of acidic mucin goblet cells (arrows). In all panels, nuclei are clearly visible by the nuclear fast red stain. <a href="https://www.jove.com/files/ftp_upload/50165/50165fig2large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs at JoVE.com

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

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

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

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

rna-situ-hybridization-whole-mount-embryos-cell-histology-adapted-for

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12.6K Views.  Union College. By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

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

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 ...

Developmental Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

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

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.

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.

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

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

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

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

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

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

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

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

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

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

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

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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

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

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

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

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

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Neuroscience

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

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

Summary

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In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

In situ Hybridization for Sipunculus nudus Coelomic Fluid