Depigmentation and Prestaining of Zebrafish Kidney: A Technique to Prepare Adult Zebrafish Kidney for Staining

When ready, use fine forceps to carefully detach the kidney from the dorsal body wall and place the organ into a glass vial. Wash the dissected kidney three times with 3 to 5 milliliters of 1x PBS with 0.05% Tween for 5 minutes each.

Next, remove the PBS solution and rinse the kidney with 3 milliliters of a 5% sucrose solution for 30 minutes. After this time, replace the solution with 3 milliliters of 30% sucrose and store overnight at 4 degrees Celsius.

The following day, remove the solution and wash the kidney two times before adding 3 to 5 milliliters of bleaching solution to remove the melanocyte pigmentation present on the kidney organ. Place the glass vial on a rotator and watch carefully as the pigmentation disappears. Depigmentation typically takes approximately 20 minutes but occasionally can take as long as 60 minutes.

If the bleaching solution is left on too long, disintegration can occur, and it is therefore advised to monitor the sample every 10 to 15 minutes to check tissue integrity. When the pigmentation has been removed from the kidney, wash twice and incubate with 4% PFA solution for 1 hour at room temperature.

Next, remove the 4% PFA solution and wash the kidney three times. After the last wash, add 3 to 5 milliliters of blocking solution and incubate the kidney at room temperature for 2 hours. When blocking is complete, proceed directly to the selected staining protocol.