Antibody Staining of Scale Melanocytes: A Technique to Detect Transcription Factors Expressed in Dorsal Scale-associated Melanocytes of Zebrafish Models

Abstract

Source: Sharanya Iyengar et al. Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model. J. Vis. Exp. (2012)

This video describes the antibody staining method to detect transcription factors expression in dorsal scale-associated melanocytes of zebrafish models. This immunostaining technique helps specifically identify the melanocyte-inducing transfection factor-A in the transgenic zebrafish.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Antibody Staining of Scale Melanocytes Make up PO4 buffer with 80 mL 0.1M Na2HPO4 and 20 mL 0.1M NaH2PO4. Make sure the pH is 7.3. Make up 2x fix buffer containing 8.0g sucrose, 0.15 mL 0.2M CaCl2, and 90 mL 0.2M PO4 buffer, pH 7.3. I…

Representative Results

Figure 1. Antibody staining of scale melanocytes. A) Scales being plucked from an anesthetized miniCoopR-EGFP zebrafish. B) Unbleached scale with pigmented melanocytes and C) bleached scale from miniCoopR-EGFP zebrafish. Scale bar = 100 μM. Unbleached scale stained with a D) Mitfa antibody and <st…

Materials

Gateway recombination reagents	Invitrogen
miniCoopR
Mitfa antibody
FITC goat anti-rabbit IgG antibody	Invitrogen
Vectashield	Vector Labs	H-1000
casper Zebrafish
701N 10 μl Syringe	Hamilton/Fisher	14-824
FBS	Invitrogen	26140079