Our in situ protocol is significant because it provides a straightforward way of visualizing gene expression patterns with minimal background staining. This technique makes a complicated and lengthy protocol relatively easy to perform. Benchtop set up suggestions and checklists provided make this protocol even easier to perform correctly and reproduce.
While the techniques shown here is specific to Astyanax mexicanus, it could likely be adapted to other systems of comparably sized individuals with small adjustments to the protocol. First, use colored lab tape to designate vials and pipettes for each gene. Using a Pasteur pipette, sort the embryos.
There are usually no more than 12 embryos per vial, once sorted. Next, set a shaking water bath to 70 degrees Celsius. Carefully draw out the methanol in the vials of sorted embryos, and replace it with 500 microliters of fresh 100%methanol.
Wash the embryos briefly for about one minute on a platform shaker. After this, rehydrate the embryos in an increasing concentration of 1x PBS with Tween 20 on a platform shaker as outlined in the text protocol. To begin, place a small gasket with a mesh bottom into the rotating water bath that has been preheated to 70 degrees Celsius.
Place aliquots of hybridization buffer minus and hybridization buffer plus into the gasket. Gently add a prepared PK solution to the vials of embryos ensuring that all tissues are completely covered in solution. Transfer the vials to a platform shaker and let them digest in the proteinase K working solution for approximately 12 minutes.
After this, gently draw off the PK solution and briefly flood the vial with PBT to dilute the remaining PK.Replace the PBT solution with 500 microliters of fresh PBT and let the solution rinse on the platform shaker for five minutes. Then replace the PBT solution with 500 microliters of thawed 4%PFA. Let the embryos incubate on the platform shaker at room temperature for 20 minutes.
To begin pre-hybridization, add 500 microliters of prewarmed hybridization buffer minus solution to the embryo-containing vials. Carefully place the vials into the gasket in the water bath at 70 degrees Celsius without shaking for five minutes. Next draw off the hybridization buffer minus solution and flood the vials with 500 microliters of prewarmed hybridization buffer plus solution.
Place the vials back into the gasket in the water bath and incubate with shaking either for four hours or overnight. To begin hybridization, draw the hybridization buffer plus out of the vials and replace it with 500 microliters of fresh prewarmed hybridization buffer plus. Carefully add two microliters of RNA probe to each vial and gently swirl to ensure even distribution of the probe.
Incubate in the water bath at 70 degrees Celsius overnight while shaking at 40 RPM. To begin, prepare microcentrifuge tubes labeled hybridization buffer plus with the gene of interest RNA probe as outlined in the text protocol. Set out two 15-milliliter conical tubes and add 0.2 grams of blocking reagent and 10 milliliters of MABT.
Place both tubes on a nutating mixer until the reagent is completely dissolved in the solution. Using a glass Pasteur pipette draw off the hybridization buffer plus and probe solution and place it into a sterile labeled microcentrifuge tube. Store this tube in the freezer at minus 20 degrees Celsius for future use.
Carefully add 500 microliters of the warm saline sodium citrate and hybridization buffer minus dilutions. Incubate in sequential solutions for 10 minutes each in the shaking water bath, and then at room temperature on a platform shaker as outlined in the text protocol. After the last incubation, replace the 100%PBT from each vial with 500 microliters of MABT.
Repeat this process twice for five minutes each. First, remove the MABT from each vial and flood it with premixed blocking solution from one of the 15-milliliter conical tubes. Place both the vials on the nutating mixer for four hours at room temperature.
Add two microliters of the antibody fragments to the second tube of prepared blocking solution and briefly vortex. Then fill each vial almost completely with blocking solution and place them on a nutating mixer overnight in a refrigerator at four degrees Celsius. To begin, prepare a stock vial of 10%NGS in MABT.
Replace the blocking solution in each vial with 500 microliters of this NGS solution. Incubate at room temperature on the platform shaker for 25 minutes. After this, replace the NGS mixture with 500 microliters of 100%MABT.
Incubate on the platform shaker at room temperature for 30 minutes. Repeat this rinse 11 more times throughout the day, every 30 minutes. Then fill each vial with 100%MABT and place them on a nutating mixer overnight in a refrigerator at four degrees Celsius.
First, replace the MABT in each vial with one milliliter of AP buffer and let it wash for five minutes. Repeat this wash twice to completely remove the MABT. Then replace the AP buffer with one milliliter of fresh AP buffer containing 3.5 microliters of BCIP and 4.5 microliters of MBT.
Replace this with a fresh mixture of AP buffer containing the BCIP and MBT every hour until the reaction is complete, making sure to monitor the reaction closely and check every 15 minutes until the desired level of staining has been achieved. Stop the coloration reaction by rinsing the embryos in an increasing concentration of 1X PBT in AP buffer as outlined in the protocol. Rinse the embryos in approximately five milliliters of 100%PBT on a nutating mixer until the desired minimum background staining is reached.
When the rinsing is complete, wash the embryos in 500 microliters of sterile PBS on a platform shaker and postfix the specimens as outlined in the text protocol. After rinsing the embryos, place them in four milliliters of 100%sterile PBS and if needed store them long-term at four degrees Celsius. In this study embryonic Astyanax specimens are labeled for high-quality gene expression analysis.
This process has been implemented successfully in both Pachon cavefish and surface fish embryos. Cavefish embryos labeled for Sox9 expression demonstrate clear labeling in the developing branchial arches and pectoral fin. Note that staining is virtually absent in the yolk sac or the developing somites on the flank.
Similarly, Tfap2a expression is evident in the portions of the developing head as early migration neural crest cells along the dorsal flank region of the embryo. The third gene presented for cavefish embryos, Phf20a, is seen in the portions of the somatic mesoderm and posterior head that are destined to give rise to bony tissue. Surface fish embryos labeled for CXCR show positive labeling in isolated regions of the head and flank, as well as a few individual cells overlying the yolk sac.
The gene Adcyap1a is expressed in regions of the central nervous system including pituitary cells. Note the highly-specific expression in paired bilateral clusters of cells on the dorsal aspect of the embryo, as well as a larger region of midline expression. The last gene examined for cavefish embryos, Sox10, is evident as an early marker of neural crest on the left and right sides of the dorsal embryo.
After completing in situ, we typically image our results using light microscopy. It's best to do this within two weeks after completion in order to avoid degradation of staining.
