We have developed a robust protocol where multiple rounds of in situ hybridization can be performed on the same drosophila brain enabling visualization of the expression patterns of many different genes. Embedding the fly brain in a hydrogel ensures excellent tissue integrity and mRNA stability over multiple rounds of hybridization. It also significantly improves optical clarity when imaging.
EASI-FISH can be adopted by any lab with a confocal or light sheet microscope to define gene expression profiles of cell types in the fly brain. To begin, Wipe a non charged slide with an RNA decontamination reagent. Remove the opaque film protecting the adhesive from the silicone gasket.
Then adhere the gasket containing up to four chambers to the slide. Use a P20 pipette to coat each chamber surface with one microliter of polylysine. Next, transfer up to four drosophila brains per tube into a 0.2 milliliter PCR tube.
Rehydrate the brains in 150 microliters of PBS containing 0.1%Triton X-100 followed by PBS. Add 40 microliters of PBS into each chamber. With a pair of fine tweezers, gently position the brains in a row at the center of the chamber, leaving some space between them.
Remove PBS from the chamber and immediately add 50 microliters of 20 millimolar MOPS buffer. While the brains are equilibrating in MOPS buffer, pipette equal volumes of thawed Melphalan-X into a tube with equal volume MOPS buffer. Then add Ac-X stock solution at a ratio of one to 100.
Vortex the solution and briefly spin it to collect it. Remove all MOPS buffer from the chambers and add 50 microliters of the Melphalan-X Ac-X solution. Place a cover slip on top of each chamber without using the gasket adhesive for sealing.
Then place the chamber in a humidified box. The next day, carefully remove the cover slip and aspirate the Melphalan-X Ac-X solution. Wash the mounted brains twice in 100 microliters of 0.1%PBT, followed by 100 microliters of PBS.
Place thawed solutions of TREx-1000 4-Hydroxy-TEMPO, TEMED, and ammonium persulfate on ice. Vortex the TREx-1000 solution to ensure that there is no precipitate. Mix the solutions to prepare a gel solution and vortex before icing.
With a pipette tip, remove the PBS from the chamber and wick away any residual liquid with a lint-free wipe. Immediately add 40 microliters of gel solution over the brains in each chamber. Incubate the chambers at four degrees Celsius for 10 minutes.
Now remove the gel solution from the chamber. Then peel off the clear protective film from the gasket surface adhesive. Add a final 45 microliters of fresh gel solution.
Gently place a cover slip over the chamber and press the cover slip to seal the chamber before incubating at four degrees Celsius for another 10 minutes. Incubate the chamber at 37 degrees Celsius for 1.5 hours to polymerize the gel. After cooling the gels on a bench, use a razor blade to carefully remove the cover slip and the silicone gasket.
Trim the gel into a rectangular shape and cut the top right hand corner to indicate sample orientation. Next, use a fine paintbrush moistened with a small amount of Pro K buffer to carefully lift the gels off the slide. Transfer each gel individually into a two milliliter microcentrifuge tube.
Mix 500 microliters of Pro K buffer and five microliters of Proteinase K enzyme, and add the mixture to each gel. Incubate at 37 degrees Celsius overnight. The next day, remove the buffer with a fine flexible pipette and then wash the gels three times in PBS for 10 minutes each.
Incubate the gels for 30 minutes in one milliliter of DNASE1 buffer at 37 degrees Celsius. Mix 50 microliters of DNASE1 enzyme with 450 microliters of DNASE1 buffer. Gently mix without vortexing.
Incubate each gel in 500 microliters of DNASE1 solution for two hours at 37 degrees Celsius. Then, wash the gels four times for 15 minutes with one milliliter of PBS at room temperature. Now, incubate the gels in 500 microliters of thawed hybridization buffer without probes for 30 minutes at 37 degrees Celsius.
Dilute the probes in hybridization buffer at a one to 100 ratio, preparing 300 microliters per gel. Incubate the gels with hybridization buffer containing probes overnight at 37 degrees Celsius. The next day, wash the gels first in probe wash buffer, then in PBS.
For the hybridization chain reaction, add 500 microliters of amplification buffer to the gel and incubate for 30 minutes at room temperature. Heat the fluorescent hairpins at 95 degrees Celsius for 90 seconds in a PCR machine and cool the solution to 25 degrees Celsius for 30 minutes. For each probe, dilute matching hairpin pairs in a one to 50 ratio in amplification buffer, preparing 300 microliters per gel.
After vortexing the mixture, add the mixture to the gels and incubate. To mount the gel for light sheet imaging, coat the gel attachment surface of a gel holder twice with one microliter of polylysine, letting it dry at 37 degrees Celsius between coats. Then, place the gel on a cover slip so that the cut is on the left hand side.
Using a paintbrush, transfer the gel onto the treated surface in one movement. Neurotransmitter associated genes and neuropeptide genes exhibited distinct expression patterns in the adult drosophila brain. VGluT and Gad1 were codetected in the same hybridization round, showing non-overlapping expression patterns.
AstA showed strong expression in the optic lobe and weaker expression in the central complex. Crz was highly expressed in the pars lateralis, while lower expression levels were observed in optic lobe neurons. Tk was detected in H delta D neurons identified with Mer-GFP expression using a specific split GAL4 line by either light sheet or confocal microscopy.
FMRFamide was absent in PFG neurons marked by Mer-GFP, but detected in surrounding neurons. High resolution imaging of FB4K neurons confirmed the spatial distribution of VGluT and ChAT/VAChT transcripts. Neuropeptides, such as AstA and Crz, are so highly expressed in some cells that individual fluorescent spots representing single transcripts can no longer be separated.
