Anti-BrdU immunohistochemistry. On the first day of BrdU immunostaining for paraffin-embedded 8 µm sections, a xylene–ethanol series is used for deparaffinization. Brains were immersed in distilled water and rinsed in a series of PBS, HCl, 8.C00.404, PBS and hydrogen peroxide PBS. Slides were incubated with mouse anti-BrdU (Sigma, Cat B8434)×200 primary antibody in 5% normal horse serum overnight. On the second day, slides were incubated with horse anti-mouse immunoglobulin G (IgG)×200 secondary antibody for 1 h. Finally, a diaminobenzidine tetrahydrochloride (DAB) reaction is performed using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA). After development, slide-mounted sections were rapidly dehydrated and cover-slipped.
