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

Overview

In this video, we demonstrate the depigmentation of adult zebrafish kidney and its subsequent preparation for staining procedures. Post-staining, the adult zebrafish kidney can be used for studying kidney regeneration, phenotypic characterization of renal disease models, and nephron functionality.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Fixation, Dissection, Permeabilization and Removal of Pigmentation of the Adult Zebrafish Kidney

1. Prepare a fresh fixative solution of 4% paraformaldehyde (PFA)/1X PBS/0.1% DMSO or thaw a frozen aliquot of 4% PFA/1X PBS and add 0.1% DMSO.
   CAUTION: PFA is toxic and PFA solutions should be handled at a chemical hood while wearing suitable personal protective equipment, including gloves and a lab coat. In addition, PFA powder should be handled with care when making the stock solution.
2. Fill a dissection tray with enough fixative solution to submerge the entire animal.
3. Euthanize and mount the selected zebrafish in fixation solution.
   NOTE: The selected fish can be an untreated zebrafish kidney specimen or a kidney isolated from a zebrafish that previously received an intraperitoneal dextran injection.
4. Fix the sample O/N (12–16 hr) at 4 ^oC.
5. The following day, use fine forceps to carefully detach the kidney from the dorsal body wall and place the organ into a glass vial using a transfer pipette.
   NOTE: Alternatively, a 12-well or 24-well culture dish can be used. While a plastic microcentrifuge tube could be used, the standard-sized tubes (1.5 mL) could restrict the washing.
6. Wash the dissected kidney 3 times with 3–5 mL of 1X PBS with 0.05% Tween for 5 min each.
7. Remove the 1X PBS with 0.05 % Tween and rinse the kidney with 3 mL of a 5% sucrose solution (in 1X PBS) for 30 min.
8. Replace with 3 mL of 30% sucrose solution (in 1X PBS) and store O/N (12–16 hr) at 4 ^oC.
   NOTE: The sucrose solutions permeabilize the cellular membranes, subsequently allowing specific labeling of reagents as they penetrate the tissue.
9. The following day, remove the 30% sucrose solution and wash the kidney 2 times with 5 mL of 1X PBS with 0.05% Tween for 5 min each.
10. Replace the 1X PBS with 0.05% Tween with 3–5 mL of bleaching solution to remove the melanocyte pigmentation present on the kidney organ.
11. Place glass vial on a rotator and watch carefully as pigmentation disappears.
    NOTE: Depigmentation typically takes approximately 20 min when performed after sucrose treatment, but occasionally can take as long as 60 min. If the bleaching solution is left on the kidney for too long, disintegration of the organ can occur; it is advised to monitor the sample every 10–15 min to check tissue integrity.
12. When the pigmentation has been removed from the kidney, wash twice with 3–5 mL of 1X PBS with 0.05% Tween.
13. Replace the 1X PBS with 0.05% Tween with 3–5 mL of 4% PFA solution for 1 hr at RT.
14. Remove the 4% PFA solution and wash the kidney 3 times with 3–5 mL of 1X PBS with 0.05% Tween for 5 min each.
    NOTE: Once fixation is complete, the kidney also can be mounted on a glass slide and evaluated for dextran uptake sans the melanocyte pigmentation.
15. Remove the 1X PBS with 0.05% Tween and replace with 3–5 mL of blocking solution, then incubate the kidney at RT in the block for 2 hr.
16. When blocking is complete, proceed directly to the selected staining protocol.
    NOTE: The kidney can now be processed through one or more other procedures in order to conduct labeling studies with different reagents.

Materials

Name	Company	Catalog Number	Comments
1X PBS			Made by diluting 10X PBS in distilled water
1X PBST			0.1% Tween-20 detergent in 1X PBS
1X PBS with 0.05 % Tween			0.05% Tween-20 detergent in 1X PBS
Tween 20	American Bioanalytical	AB02038
4% PFA/1X PBS			Dissolve 4% PFA (w/v) (Electron Microscopy Sciences, Cat #19210) in 1X PBS, bring to boil on a hot plate in a fume hood. Cool and freeze the aliquots for storage in the freezer at -20°C. Thaw just before use and do not refreeze stocks
Bleaching solution			Bleach mix formula (for a 20 ml solution): 1.6 ml of 10% Potassium hydroxide solution 0.6 ml of 30% Hydrogen peroxide solution 100 μl of 20% Tween. Fill to 20 ml with distilled water.
Potassium hydroxide	Sigma	221473
Hydrogen peroxide	Sigma	H1009
Blocking solution			Blocking Solution (for a 10 ml solution): 8 ml of 1X PBS with 0.05% Tween 2 ml of fetal calf serum 150 μl of DMSO