Immunofluorescent Labeling of Zebrafish Larvae

To detect GFP, phospho-Paxillin, Vinculin or Myogenin, larvae were anesthesized in 0.02% MS-222, fixed in 4% paraformaldehyde (PFA, Sigma) overnight at 4°C and subsequently stored in 100% methanol (Fisher) at −20°C. Samples were washed in 0.1% Tween20 in phosphate-buffered saline (PBT, Sigma) and permeabilized. Samples were blocked in 5% goat serum (Life Technologies) in PBT for at least 2 h, then incubated with primary antibody diluted in 5% goat serum/PBT over night at 4°C. Samples were washed for at least 4 h in 0.1% PBT the following day, then incubated with secondary antibodies diluted in 5% goat serum/PBT for 2 h at room temperature. After several washes in PBT, samples were taken through glycerol series and mounted in VectaShield with DAPI (Vector Laboratories).
To label dividing cells with BrdU, larvae were exposed to 10 mM BrdU diluted into E3 medium and processed as previously described [16 (link)].
Antibodies used were chicken polyclonal anti-GFP (1 : 500; Millipore), rabbit polyclonal anti-GFP (1 : 500; Life Technologies), rabbit polyclonal anti-phospho-Paxillin (Tyr118) (1 : 200; Thermo Scientific), mouse anti-Vinculin (1 : 200; Sigma), rabbit polyclonal anti-Myogenin (1 : 50; Santa Cruz Biotechnology), rat monoclonal anti-BrdU (1 : 250; Abcam). Secondary antibodies used were Alexa conjugated antibodies diluted 1 : 500 in goat serum and PBT.