Immunofluorescent Labeling of Zebrafish Brains

Larvae were fixed in 4% para-formaldehyde/PBS overnight at 4 °C. They were then rinsed in PBS. The brains were dissected out, and permeabilized using 1% BSA (fraction V; Sigma), 0.1% DMSO and 0.1% Triton X-100. The anti-GAD65/67 (Abcam ab11070, RRID:AB_297722; 1:500) has been previously used in zebrafish [70 (link)]. The brains were incubated in the primary antibody overnight, rinsed several times in PBS, then incubated in secondary antibody (Alexa 488 goat anti-rabbit; 1:1000). After washing, these were mounted in 1.2% agarose/PBS. Imaging was carried out using a Zeiss LSM 800 laser scanning confocal microscope, with a 40× water immersion objective.