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

Abstract

Source: McCampbell, K. K. et al. Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney. J. Vis. Exp. (2014)

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 handle....

Materials

List of materials used in this article

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