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

Overview

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

1. 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. If necessary, adjust the pH to 7.3 with NaOH or HCl, then make up to 100 ml with PO₄ buffer.
2. Weigh up to 300 mg solid PFA and add to a microcentrifuge tube. Add 5 mM NaOH to a volume equal to 4.5 x mass, in mg, of PFA (e.g. 450 ul 5 mM NaOH to 100 mg PFA). Heat at 60-70 °C with occasional shaking until the PFA is dissolved. Spin down remaining particulates and recover 20% PFA supernatant. Make it fresh every time.
3. Prepare fixation solution containing 1x fix buffer and 4% PFA.
4. Anesthetize the fish in 0.17 mg/mL tricaine.
5. Using incident light under a dissecting microscope, pluck dorsal scales using sharp fine-tipped forceps, taking care not to damage the melanocyte-containing half of the scale (Figure 1A). Place the scales directly from the forceps into 1 mL fixation solution and incubate at room temperature, while turning, for ≥ 2 hr. Transfer the fish into fish water and monitor the fish to confirm successful recovery.
6. To bleach pigmented melanocytes, wash fixed scales in PBST (1x PBS pH 7.4, 0.1% Triton X-100) 2 times for 5 min at room temperature. Add 1 ml freshly made bleach solution containing 0.4 mL 10% KOH and 0.15 mL 30% H₂O₂ in 5 mL with dH₂O. Put on parafilm or cap locks to prevent gas from bursting the tubes open. Continue bleaching until pigment is gone (10 min is typical) (Figure 1B, C). Wash in PBST four times for 5 min at room temperature.
7. Block for at least 30 min in block solution made up of 1x PBS pH 7.4, 0.2% Triton X-100, 2 mg/mL BSA, 1% DMSO, 0.02% NaN₃, and 2% lamb serum. Leave out NaN₃ if developing with HRP reaction.
8. Incubate with primary antibody in block solution overnight at room temperature. You need to have at least 400 μL of solution over the samples to keep them submerged.
9. Wash the scales 3 times for 30 min in PBST at room temperature.
10. Incubate with secondary antibody from 2 hr to overnight in block solution at room temperature.
11. Stain for 10 min with PBST + 0.1 μM DAPI.
12. Wash 3 times for 5 min with PBST.
13. Mount the scales on a glass slide in a drop of vectashield so that the concave side of the scales faces down towards the slide and place a glass cover slip over them. Seal the edges of the slide with clear nail polish and observe the slides under a fluorescence microscope (Figure 1D, E, F, G).

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 E) DAPI. Bleached scale stained with a F) Mitfa antibody and G) DAPI. Scale bar = 40 μM.

Materials

Name	Company	Catalog Number	Comments
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