Immunohistochemistry of Nervous System

Animals were fixed with AB fix [4% Paraformaldehyde and 1×PBST (1% TritonX)] for 3 h at 23°C and then washed in 1×PBST (5% TritonX), ddH20Tx (5% TritonX), and acetone for 5 min each at 23°C and then an additional acetone wash at −20°C. 1×PBST (5% TritonX) with 5% goat serum was used to block for at least an hour. Animals were incubated in primary antibody overnight at 4°C. The primary antibodies used in this study include the following: Sox10, 1∶5,000 [49] (link); Acetylated Tubulin, 1∶10,000 (Sigma); HuC, 1∶100 (Invitrogen); MBP, 1∶250 [27] (link). Animals were washed extensively with 1×PBSTx before the secondary antibody was added. These antibodies include Alexa antibodies (1∶600): goat anti-rabbit 568, goat anti-mouse 568, goat anti-rabbit 647, and goat anti-mouse 647. After extensive washes, animals were stored in 50% glycerol/50% 1×PBS until imaged and mounted under a bridged coverslip.