Brains were dissected in PBS and fixed in 4%PFA + 0.5% Triton X-100 (Triton) at RT for 20min, nutating. Brains were rinsed 2x with PBST (0.5% Triton X-100) at RT, then washed 1x with 0.5% PBSTriton at RT for 30min, nutating. For primary antibody staining, brains were incubated in Starting Block + 0.5% Triton at RT for 30min. Antibodies were then added and the brains were incubated at 4C overnight, nutating. Brains were then washed as described above followed by incubating in Starting Block + 0.5% Triton at RT for 30min. Secondary antibodies were added and brains were incubated at RT for 2hr, protected from light. Brains were finally rinsed 2X in PBST (0.5% Triton X-100) at RT for 1min each, then washed 2X in PBS for 30min. Brains were subsequently mounted as described in the HCR section above.
Antibodies were diluted as the following: Mouse-anti-Fas3 1:50 (DHSB), Rat-anti-dpn (1:1000) (C-YL lab), Donkey-anti-Mouse (AF488) 1:500 (Jackson ImmunoResearch Laboratories, Inc.), Donkey-anti-Rat (AF647) 1:500 (Jackson ImmunoResearch Laboratories, Inc.).
Antibodies were diluted as the following: Mouse-anti-Fas3 1:50 (DHSB), Rat-anti-dpn (1:1000) (C-YL lab), Donkey-anti-Mouse (AF488) 1:500 (Jackson ImmunoResearch Laboratories, Inc.), Donkey-anti-Rat (AF647) 1:500 (Jackson ImmunoResearch Laboratories, Inc.).