Depigmentation and Prestaining of Zebrafish Kidney: A Technique to Prepare Adult Zebrafish Kidney for Staining
Published: April 30, 2023
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 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…
Divulgaciones
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 |
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Depigmentation and Prestaining of Zebrafish Kidney: A Technique to Prepare Adult Zebrafish Kidney for Staining. J. Vis. Exp. (Pending Publication), e20574, doi: (2023).