In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

This protocol is significant because it allows the determination of gene expression in older zebrafish larvae and juveniles during metamorphosis. The advantage of this technique is that several steps have been optimized for probe penetration and visualization of the kidney. To begin, set up adult zebrafish to mate by adding one male and one female fish in a mating tank in the late afternoon after their last meal.

The next day, collect the embryos in Petri plates containing E3 medium. Five days post-fertilization, place a 400-micrometer screen in a 2.8-liter tank and fill it with 2 centimeters of system water. Then add the larva from one Petri plate.

To fix the larva, remove a tank of larva at the desired time point, then, using a net and transfer pipette with its tip cut off, transfer the larva to the Petri plate. Next, add two milliliters of 2%tricaine to immobilize the larva. After immobilization, replace the tricaine with 20 milliliters of fixing solution.

After 30 minutes, transfer the larva into a 50-milliliter tube containing 25 milliliters of fresh fixing solution. Then, ensuring that the cap is tight, slowly rock the tube at four degrees Celsius for two days. On day three, replace the fixing solution with 20 milliliters of PBST then transfer the larva to a Petri plate.

To measure the larva, place the Petri plate on top of a flat ruler under a dissecting microscope, then, using an eyelash manipulator, move each larva onto the ruler to measure its total length. After measuring all larva, combine several larva of similar lengths onto one 5.5-milliliter glass vial with PBST. For dehydration, replace the PBST in the glass vial with four milliliters of 100%methanol, then store the vial at minus 20 degrees Celsius for two days.

For rehydration, replace the 100%methanol with four milliliters of a 75%methanol and 25%PBST solution and rock the vial for five minutes. Then, replace the methanol and PBST solution with four milliliters of fresh PBST and rock the vial again for 10 minutes. For Proteinase K digestion, replace the PBST with two milliliters of a Proteinase K solution and rock the vial at room temperature for 30 minutes.

Next, for bleaching, transfer the larva to a six-well plate and replace the PBST with three milliliters of fresh bleaching solution. After the complete disappearance of pigmentation along the mesonephros, transfer the larva back into a glass vial, replace the bleaching solution with four milliliters of PBST, and rock the vial for 10 minutes. For pre-hybridization, replace the fixing solution with four milliliters of PBST and rock the vial for 10 minutes, then, replace the PBST with four milliliters of Hyb-solution and rock for 10 minutes.

After two additional incubations in the Hyb-solution, replace the solution with four milliliters of the Hyb+solution. Simultaneously dilute the EGFP fluorescein probe one to 100 in 500 microliters of hybridization plus solution, and incubate the vial and probe at 70 degrees Celsius overnight. The next day, to hybridize the probe, replace the Hyb+solution in the vial with the preheated probe and incubate at 70 degrees Celsius overnight.

The following day, add 50 milliliters of preheated 0.2 times SSCT into a 50-milliliter tube. Then, insert a 100-micrometer cell strainer into the top of the tube, transfer the larva from the glass vial into the cell strainer, and ensuring that the larva are submerged in the buffer, incubate the vial at 70 degrees Celsius for two hours. After the incubation, transfer the cell strainer into a new tube containing preheated 0.2 times SSCT, and incubate again at 70 degrees Celsius for two hours.

Next, for blocking, transfer the larva to a new glass vial and allow it to cool to room temperature. Then, replace the 0.2 times SSCT solution with four milliliters of a 67%0.2 times SSCT and 33%MABT solution and rock the vial at room temperature for 10 minutes. Next, replace the SSCTT MABT solution with four milliliters of fresh MABT, and incubate the vial on a rocker for 10 minutes.

Then, replace the MABT with four milliliters of blocking solution and incubate four degrees Celsius overnight. The next day, replace the blocking solution with an antibody solution and incubate at four degree Celsius for two days. For antibody washing, transfer the larva into a 50-milliliter tube, then add 40 milliliters of PBST2, and lay the tube laying on its side for overnight incubation at four degrees Celsius.

On day 12, after transferring the lava to a six-well plate, replace the PBST2 with three milliliters of staining buffer. After a five-minute incubation on a rocker, replace the staining buffer with three milliliters of the staining solution. When the desired staining intensity is reached, replace the staining solution with three milliliters of the stopping solution and rock for 30 minutes.

Then, transfer the larva to a new glass vial, replace the stopping solution with four milliliters of fresh fixing solution, and incubate for one hour at room temperature. For imaging, replace the fixing solution with four milliliters of fresh PBST. Following a 10 minute incubation on a rocker, transfer the larva to a six-well plate, then replace the PBST with four milliliters of fresh PBST and rock the plate again for 10 minutes.

Next, add three milliliters of 50%glycerol in PBST and rock for 10 minutes, then, using a dissecting microscope, image the larva directly in the six-well plate. This in situ hybridization protocol effectively labels kidney progenitor cells and various nephron structures using mesonephros development. The initial mesonephric nephron forms at approximately 5.2 millimeters dorsal to the pronephros.

Clusters of progenitor cells are present during mesonephros development in addition to single progenitor cells. In larger juveniles, background staining can occur in the somites. Overall, this method can be used to study other tissues that form during metamorphosis, in addition to dissected adult organs.