CD24 Antibody (M1/69): sc-19651

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Perfused brains were sectioned coronally at 35 μm intervals using a sledge microtome (Leica SM 1400) and collected in 6 series in antifreeze-based solution. Immunohistochemistry was performed as previously described [23]. Briefly, free-floating sections were washed in 0.1M phosphate buffer saline (PBS) before being quenched for 20–30 min in 3% H2O2 and 50% methanol. The sections were then washed in PBS again, before being incubated overnight in 5% normal horse serum plus 0.05% Triton X-100 in PBS at room temperature with a primary antibody recognizing Rat-CD24 (M1/69; sc-19651, 1:50; Santa Cruz), rat-dopamine transporter (DAT; 1:200; MAB369, Chemicon), Goat-GDNF (1:500; AF-212-NA; R&D systems), and Rabbit-TH (1:1000, Pelfreez). A one-hour incubation with an appropriate biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) was followed by an additional 40-minute incubation in avidin-biotin-peroxidase solution (ABC Elite; Vector Laboratories). Finally, visualization of the bound antibody was done using 3'-diaminobenzidine (DAB; Sigma). The sections were then mounted on gelatin-coated glass slides, dehydrated in escalating ethanol concentrations, cleared in xylene, and cover slipped with DEPEX Mounting Medium.

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