In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

This method can help answer key questions in coral cell biology, such as what types of tissues and cells express specific types of genes. The main advantage of this technique is that you have visual information on RNA expression. Though this method can provide insight into spatial gene expression in adult corals, it can also be used in other life stages of corals, as well as in other invertebrate systems.

Generally individuals new to this method will struggle because there's so many steps and solutions to keep track of. So they need to pay attention to where they are in the protocol, and keep good track of time when doing the washes. Kevin Rodriguez, a technician in my laboratory, will be demonstrating this procedure.

To begin, dewax thin sectioned paraffin embedded slides with 100%xylene in glass Coplin jars for 10 minutes under the hood. Next, fill four sterile glass Coplin jars with 100%ethanol, 80%ethanol, 70%ethanol and 60%ethanol respectively. First, transfer the slides to the Coplin jar containing 100%ethanol, and allow them to soak for 10 minutes.

Then transfer the slides to another Coplin jar containing 100%ethanol. Repeat this process with each of the Coplin jars in decreasing order of ethanol concentration. First transfer the slides to a sterile slide mailer with 18 mL of PBS.

Then wash the slides for five minutes at room temperature. Next, pour off 1 volume of PBS, and add approximately 10 mL of proteinase K solution to the mailer. Then incubate the mailer at 37 degrees Celsius for 15 minutes.

While the slides incubate, place prehyb buffer in a boiling water bath for 10 minutes. Then allow the buffer to cool in an ice bath for 5 minutes. To stop digestion by the proteinase K reaction, remove the proteinase K and add 18 mL of 0.2%glycine PBS solution to the mailer.

Then allow the mailer to sit at room temperature for 5 minutes. After this, remove the glycine PBS solution and replace it with 18 mL of 2x saline sodium citrate, or SSC. Wash the slides in SSC for 10 minutes at room temperature with shaking at 100-150 RPM.

While the slides incubate in the hybridization oven, dilute the probes in hybridization buffer. Then place the diluted probes on a heat block at 86-90 degrees Celsius for 12 minutes, and allow them to cool on ice for one minute. Next, remove the slide mailer from the hybridization oven, and remove each slide with sterile tweezers.

Lay the slides on a paper towel, and carefully remove excess buffer around the tissue samples. Using a PAP pen, draw a circle around each tissue sample. Then use a pipette to apply 25 microliters of diluted probe solution, and cover each sample with a plastic coverslip.

After this, add 4x SSC and 50%formamide solution in the bottom of a slide moisture chamber. Then place the slides in the moisture chamber. Wash the slides with 18 mL of alkaline phosphatase buffer without magnesium chloride for 1 minute.

Then pour out the buffer and replace it with 18 mL of Boehringer Mannheim blocking buffer diluted with maleic acid buffer. Incubate the slide mailer overnight at room temperature with gentle shaking. Next, prepare 20 mL of diluted DIG anti-deoxigenin-AP Fab fragments.

Add the anti-dig antibody to a new sterile slide mailer, and transfer the slides from the old mailer. Then incubate the slides at room temperature for three hours with gentle shaking. After the incubation period, pour the anti-dig antibody out, and wash the slides with 18 mL of AP buffer without magnesium chloride for five minutes with gentle shaking.

Next, pour out the AP buffer without magnesium chloride and add 18 mL of AP buffer. Wash the slides for five minutes with gentle shaking. Then remove the old AP buffer, replace it with 18 mL of fresh AP buffer, and repeat the wash.

In a dark room, pour off the AP buffer and add 18 mL of BM-Purple to the mailer. Then incubate the slides at room temperature, checking for purple color development every half hour. To stop color development, transfer the slides to a new sterile mailer with 18 mL of TE buffer.

Allow the slides to sit in the dark at room temperature for five minutes. Next, pour off the TE buffer, add 18 mL of RNase-Free water to the mailer, and wash the slides for one minute. Then remove the slides from the water and dry them with tissue.

Add glycerol mounting medium, and place a coverslip on each slide. Finally, store the slides at 4 degrees Celsius until pictures are ready to be taken. Using this protocol, heat stressed coral tissue expressing AP-1, FosB, and TNFR41 were identified.

As indicated by the diffuse tissue staining, the expression of anti-sense AP-1 is found throughout the epidermis and oral gastrodermis. Cell specific staining was also observed in FosB and TNFR41 expression. FosB stained cnidocytes and was specific to both spirocytes, which produce spirocysts, and nematocytes, which produce the microbasic mastigophore organelle.

Likewise, TNFR41 was also expressed in nematocytes and spirocytes. While attempting this procedure, it's important to remember to not rush anything, because the washes and the incubations take time.