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 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 hand…
Divulgazioni
The authors have nothing to disclose.
Materials
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
Depigmentation and Prestaining of Zebrafish Kidney: A Technique to Prepare Adult Zebrafish Kidney for Staining. J. Vis. Exp. (Pending Publication), e20574, doi: (2023).