Alexa fluor dyes

Cortical sections (15 μm) were harvested using a freezing microtome (Leica CM1950). During immunohistochemistry, the brain sections were washed with 1% PBST (1% Triton X-100 in 1 M PBS [pH 7.4]), fixed for 30 min by 4% PFA, and washed by PBST for 15 min three times. The slices were incubated with blocking buffer (5% BSA in 1% PBST) for 1 hr at room temperature. The slices were incubated in primary antibodies at 4°C overnight and then washed with PBST for 15 min three times. The secondary antibodies conjugated with Alexa Fluor dyes (1:1,000 dilution; Jackson ImmunoResearch) were added for 1 hr at room temperature. The brain sections were washed three times for 15 min in PBST, incubated with 2 mg/mL DAPI (Sigma, D9542) for 2 min, and washed three times for 5 min with PBST. When IHC was required for BrdU, the slices were incubated in ice-cold 1 M HCl for 10 min, 2 M HCl for 10 min at room temperature, and 2 M HCl for 20 min at 37°C, then washed with PBST three times before blocking and anti-BrdU antibody incubation.
Immunostaining for cultured cells was performed according to the following procedure: the cells were washed with PBST (0.1% Triton X-100 in PBS), fixed in 4% PFA, blocked by 5% BSA, incubated with primary antibodies overnight at 4°C, and visualized using fluorescence-labeling secondary antibodies.