Zebrafish Immunohistochemistry for Labeling Proteins

Zebrafish embryos and larvae were fixed in 4% PFA/1x PBS at 4° C overnight, and then incubated with 150 mM Tris-HCl, pH 9.0 at 70° C for 15 minutes for antigen retrieval^{52 (link)}. Samples were then permeabilized with 100% acetone at −20° C for 20 minutes, and antibody staining was performed via standard procedures^{50 (link)}. The following primary antibodies used: chicken α-GFP (Abcam, 1:1000), rabbit α-RFP (Rockland, 1:1000), mouse α-SV2 (DSHB, 1:200), and mouse α-GS (Sigma, 1:1 prediluted). The following secondary antibodies used: Alexa 488 α-chicken (Jackson ImmunoResearch, 1:250), Rhodamine Red-X α-rabbit (Jackson ImmunoResearch, 1:250), and Dylight 649 α-mouse (Vector Laboratories, 1:250). Stained samples were mounted in Vectashield antifade mounting medium (Vector Laboratories) for confocal analysis.