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 synthe...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <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></td>
 <td></td>
 <td></td>
 <td>Table 3. Reagents and equipment for RNA whole mount in situ's.</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></td>
 <td></td>
 <td></td>
 <td>Table 4. Reagents and equipment for Alcian Blue stain.</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 dista...

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

Research

JoVE Journal

Biology

12.6K visualizações.  Union College. Através da combinação de métodos para a montagem de toda RNA In situ Hibridação e histologia, a expressão do gene pode estar relacionada com as decisões celulares destino no embrião em desenvolvimento. Estes métodos têm sido adaptados para elasmobrânquios marinhos e facilitar o uso desses animais como organismos-modelo para estudos de toxicologia, biomédicas e comparativo.

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

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

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

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

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

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

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

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

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

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

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

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

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

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

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

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

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol 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 ...