The overall goal of this procedure is to monitor gene expression and sulfate decisions in a developing marina brink. This is accomplished by first hybridizing, a gene specific RNA probe in home mounted embryos. The second step of the procedure is to visualize gene expression in the embryos after sectioning and staining for Sian blue and nuclear fast red examine the tissue for the expression of gene specific messages and the presence of acid mucin producing goblet cells.
Hello, I'm Nicole the Dosio, and I'm an assistant professor of biology at Union College. The main advantage of this technique over existing methods for RNA whole mount and situ hybridization is that this protocol is specifically adapted for marine SMA ranks. Begin this protocol with staged skate embryos in methanol stored at minus 20 degrees Celsius, and a prepared RNA probe for embryos staged.
At 29 30 or later, an epidermis body may be lifted from the embryo under the microscope. Carefully dissect off the epidermal layer to maximize the probes penetration. The most critical step of this protocol is ensuring complete penetration of the probe for older embryos.
This includes removing the outer epidermal layer from embryos and adequate proteinase K treatment. Transfer the embryos in clean glass scintillation vials and rehydrate them using a reverse methanol series with gentle rocking in each solution for five to 30 minutes at room temperature, complete the rehydration with two washes in PBT for 10 minutes each with gentle rocking bleach and 6%hydrogen peroxide PBT for one hour rocking. Then repeat the PBT wash with rocking.
Next, treat the embryos with PK for penetration of the probe during hybridization. The duration of the incubation and the amount of enzyme used must be determined empirically. It is best used fresh PK enzyme that has been titered and tested.
Next, quickly rinse the embryos in PBT to wash away the pk. Then to deactivate the PK postfix the embryos with 4%PFA 0.2%glutaraldehyde in PBT for 20 minutes. With gentle rocking, remove the PFA solution with two washes and PBT of 10 minutes each.
For pre hybridization, add just enough preheated hybridization solution to cover the embryos, typically two to three milliliters. Then incubate the embryos at 70 degrees Celsius for one hour with gentle agitation. To prepare the hybridization solution, preheat the RNA probe to 70 degrees Celsius for 10 minutes and let it cool on ice.
We're replace the pre hybridization solution with fresh preheated hybridization solution just enough to cover the embryos at 15 to 20 microliters of the RNA probe to the vial. Tightly secure the lid and set it rocking gently at 70 degrees Celsius overnight. Continue the protocol by removing the probe solution from the embryos in the vial.
Then follow a series of six washes through solution one and solution two, all with gentle rocking. Transfer the embryos to a net well in a six well tissue culture plate, followed by three washes with TBST to pre block the embryos. Prepare a solution of 10%heat inactivated sheep serum in TBST.
After adding the solution to the plate, rock the embryos at room temperature for one hour to add antibodies. Transfer the embryos from the net well to a glass sation vial containing one to 5, 000 anti DIG fab fragments in 1%heat and activated sheep serum TBST. Set them rocking overnight at four degrees Celsius the next day.
In the morning, remove the antibodies and place the embryos back into the net wells over the course of the day. Perform a series of washes in TBST and finish with an overnight wash. Once the antibody washes are complete, transfer the embryos to a glass insulation vial with fresh NTMT.
Wash them three times in fresh NTMT for 10 minutes per wash. Replace the last NTMT wash with an NBT plus BCIP reaction mixture. Because these reactants are light sensitive.
Cover the sation vial in foil, then set to rock at room temperature. Monitor the color reaction every 15 minutes until the desired gene expression is clearly visible When the reaction is complete, wash the embryos twice in PBT, then postfix the embryos in 4%paraform aldehyde plus 0.1%glu aldehyde for an hour. Finally, visualize the embryos with a dissecting stereo microscope.
After embedding and sectioning the EMA brank tissue, place the slides with the sections facing upward on a slide warmer. Melt them for 45 minutes. Next, take the slides in a slide holder to a fume hood.
Dissolve the paraffin by washing the slides twice in xylene for five minutes per wash. Then rehydrate the slides in an ethanol series at one minute per solution. Now stain the rehydrated tissue in allium blue solution pH 2.5 for 15 minutes.
Develop the stain by rinsing the tissue in running tap water for three minutes. Then dip the rinse tissues once and distilled water and put them into the nuclear fast red counter stain solution for 10 minutes. Again, rinse the slides and running tap water for three minutes and dip them in distilled water.
Now dehydrate the tissue in an ethanol series using 20 dips per solution. After the dehydration, wash the slides in xylene twice for five minutes per wash. Mount the prepared tissues with DPX mounting medium and cover slips before manipulating slides.
Allow the mounting medium to dry and harden for 48 hours in the hood. RNA hole mount in C twos of skate embryos depict expression of sonic hedgehog and hox. Eight 13 expression of sonic hedgehog and higher vertebrates is found in the not cord and gut endoderm, and this expression pattern is conserved in the skate marine sma.
Brinks have a unique method of osmo regulation that uses the rectal gland to secrete salts hox. A 13 expression is high in the developing rectal gland, but its role here remains unknown. Staining the digestive tract with alium blue reveals acid mucin containing cot cells.
The distribution of acid mucins differs throughout the digestive tract reflecting differences in acid mucin function. Acidic mucins are sparsely produced in the spiral intestine and C cloaca, whereas in the distal intestine, they are at a higher concentration. While attempting this procedure, it's important to follow common practices for handling RNA in order to avoid contamination by nucleases.
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