Immunohistochemistry was performed as previously described20 (link). Antibodies used were: mouse monoclonal anti-GFP 1:250 (Millipore), rabbit anti-synapsin 1:1000 (Synaptic Systems)22 (link), rabbit anti-PSD95 (Abcam)23 (link), Cy-3 anti-rabbit 1:400, Alexa 488 donkey anti-mouse 1:400 (Invitrogen), and rabbit anti-goat Alexa 555 (Invitrogen). Double immunohistochemistry for GFP with anti-synapsin or anti-PSD95 was performed at 3 dpf as follows: embryos were fixed in 4% paraformaldehyde (PFA) in PBS for 1.5 hours at room temperature (RT) and washed briefly in PBS (3 × 5 min) with 0.1% Triton X-100 (PBST). Embryos were then blocked (PBS with 1% BSA, 1% DMSO, 2% goat serum, and 0.1% Triton X-100) for 3 hours at RT, and then incubated in blocking buffer with primary antibodies overnight at 4 °C. Embryos were washed with PBST, and incubated with secondary antibodies overnight at 4 °C.
For sectioning, stained larvae was cryoprotected in 30% of sucrose with PBS for at least 2 hr, rinsed briefly in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA), and frozen in a Tissue-Tek O.C. ethanol/dry ice bath. Sections were 20 μm and mounted on the Superfrost Plus Microscope slides.
For sectioning, stained larvae was cryoprotected in 30% of sucrose with PBS for at least 2 hr, rinsed briefly in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA), and frozen in a Tissue-Tek O.C. ethanol/dry ice bath. Sections were 20 μm and mounted on the Superfrost Plus Microscope slides.