Immunohistochemistry and Cilia Labeling in Fish

Fish used for immunohistochemistry were fixed for 2 hr at room temperature in 4% paraformaldehyde. Antibody labeling was carried out as previously described (Stawicki et al. 2014 (link)). Fish used for hair cell counts were labeled with a mouse anti-parvalbumin primary antibody (Millipore, MAB1572) diluted at 1:500 in antibody block (5% heat-inactivated goat serum in PBS, 0.2% Triton, 1% DMSO, and 0.2% BSA). Fish used for the analysis of cilia morphology were labeled with a mouse anti-acetylated tubulin primary antibody (Sigma-Aldrich, 6-11B-1) diluted at 1:1000 in antibody block. For phalloidin labeling, after fixation fish were incubated for 2 hr at room temperature in Alexa Fluor 488 Phalloidin (Molecular Probes) diluted 1:100 in antibody block. They were then washed two times in PBST, one time in PBS, and stored in a 50:50 PBS and glycerol mixture prior to use.