In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol use a combination of in situ hybridization and the immunohistochemistry, and it serves as an easy and efficient way to simultaneously analyze one mRNA and one protein. This protocol is applicable to embryos at different stages, tissue sections of various thickness, and different organs. To begin, fix approximately 10 embryos with 0.5 to 1 milliliter of fresh 4%paraformaldehyde in a 1.5 milliliter microfuge tube at four degrees Celsius overnight for 12 to 14 hours.

Gradually dehydrate the embryos by washing with 25, 50, and 75%methanol in PBST successively for five minutes each at room temperature. Then, wash the embryos in 100%methanol for five minutes at room temperature. Incubate the embryos in 100%methanol at minus 20 degrees Celsius for at least overnight, for 12 to 14 hours.

Gradually rehydrate the embryos by washing with 75, 50, and 25%methanol in PBST successively for five minutes each at room temperature. Again, wash the embryo thrice with PBST for five minutes each at room temperature. Digest the embryos with 10 micrograms per milliliter of proteinase K in PBST at room temperature, and wash the embryos thrice with PBST for five minutes.

Then, refix the washed embryos in 4%paraformaldehyde for 15 minutes at room temperature. Again, wash the embryo thrice with PBST while incubating for five minutes during each wash. For pre-hybridizing the embryos, incubate them in pre-hybridization solution at 65 degrees Celsius for five minutes.

Then, replace the pre-hybridization solution with the hybridization solution and pre-hybridize for at least four hours. Heat the probe in the hybridization solution for five minutes at 95 degrees Celsius before adding to the embryos. Remove as much pre-hybridization solution as possible without letting the embryos come into contact with the air.

To the tube containing the embryos, add preheated probe in the hybridization solution and allow the probe to hybridize overnight for 12 to 14 hours at 50 to 70 degrees Celsius. On the next day, aspirate the probe solution with a pipette and store it in a tube at minus 20 degrees Celsius so that it can be reused many times. Wash the embryos with 100%hybridization solution followed by sequential washing with 75, 50, and 25%hybridization solution in twice the concentration of SSCT for 15 minutes at 65 degrees Celsius.

Then, wash the embryos in twice and 0.2 times the concentration of SSCT for 15 minutes at 65 degrees Celsius. Wash the embryos twice for 10 minutes in MABT at room temperature. Block the hybridized and washed embryos for at least two hours at room temperature, with 2%blocking solution-1.

Replace the blocking solution-1 with antidigoxigenin alkaline phosphatase in a fresh 2%blocking solution-1 and shake overnight for 12 to 14 hours at four degrees Celsius. Then, wash the embryos four times in MABT for 30 minutes at room temperature. Again, wash the embryos twice in NTMT and remove as much NTMT as possible from the embryos using a pipette.

Replace with BM purple alkaline phosphatase substrate and stain the embryos at room temperature in the dark. Once the stain has developed, stop the reaction by briefly rinsing twice with NTMT. After in situ hybridization, rinse the embryos with PBST thrice for 20 minutes.

Immerse the embryos in 5%sucrose in PBS overnight for 12 to 14 hours at four degrees Celsius. Then, change the solution covering the embryos to 15%sucrose in PBS and incubate overnight for 12 to 16 hours at four degrees Celsius. Again, change the solution covering the embryos to 30%sucrose in PBS and incubate for one to two days at four degrees Celsius.

Transfer the embryos to a new cryomold for tissue and gently fill it with optimal cutting temperature medium while avoiding the formation of bubbles. Next, cut the specimens into 12 to 20 micrometer thick sections using a cryostat and quickly transfer the sections to glass slides. Allow the samples to reach room temperature and store the sections in a sealed slide box at minus 80 degrees Celsius for subsequent use.

Wash the slides containing the sections with PBS for five minutes. Then, place the slides in the buffer and heat the solution for approximately 20 minutes to keep it near boiling temperature. Drain the excess solution and carefully dry the area around each section with a piece of tissue.

Then, draw a circle around the section with a water-repellent pen to form a hydrophobic barrier and block the section for two hours in blocking solution-2 at room temperature. Pipette primary antibody solution per slide and incubate the slides in an immunohistochemical wet box at four degrees Celsius overnight. Wash the slides three times with PBS while incubating for 10 minutes during each wash, and drain the excess PBS.

Then, incubate the slides with the appropriate secondary antibody for one hour at room temperature in PBS. Again, wash the slides thrice with PBS while incubating for 10 minutes during each wash, and drain the excess PBS. Subsequently, pipette the mounting medium onto the slide and mount with a slide coverslip.

The expression of 5-HT2C receptor in the central nervous system was detected in the transgenic foxP2 egfp-caax line. The simultaneous expression of 5-HT2C was observed in the transgenic line together with foxP2 neurons, which were labeled by fluorescein green fluorescent protein. The expression of insm1a, a zinc-finger transcription factor in the zebrafish spinal cord and motor neurons, was detected in the transgenic line.

The simultaneous expression of insm1a was observed in the transgenic line together with hb9 neurons, which were labeled by fluorescein green fluorescent protein. Zebrafish must be fixed with freshly-prepared paraformaldehyde.