Immunostaining of Adult Brain Tissue

Adult brains were dissected in 1 × PBS containing 0.05% Triton-X 100 (PBT), and then fixed overnight at 4° in 2% paraformaldehyde (EM Sciences) in PBT. They were washed for 5 × 10 min in 0.1% PBT, blocked for 1 hr in 0.1% PBT with 0.5% BSA and 5% normal goat serum, and then incubated with primary antibodies overnight at 4°. They were then washed, blocked, and incubated with secondary antibodies overnight at 4°. Washed tissues were mounted on glass slides in Vectashield (Vector Laboratories), small pieces of broken coverslips serving as posts, covered with a coverslip, and sealed. Primary antibodies were rabbit anti-DopR (1:1250, Kong et al. 2010 (link)), mouse anti-CD2 (1:50, Pierce), and mouse anti-Cherry (1:200, Biorbyt). Secondary antibodies were goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 (Life Technologies). The tissues were imaged on a Nikon Eclipse Ti C1 Confocal System using 1 μm steps and a 40 × or 60 × oil-immersion lens.