Immunocytochemistry on brain sections and neurospheres

Immunohistochemistry of SVZ and OB was performed on 40-μm serial free-floating sections. To improve the efficiency of BrdU detection, sections and cells, prior to antibody staining, were exposed to 2 N HCl for 30 min (for sections) or 15 min (for cells), respectively, at 37 °C and then washed with 0.1 M sodium borate buffer pH 8.5 for 10 min (to allow the denaturation of DNA, necessary to expose the BrdU). Upon fixation, sections or cells were permeabilized in blocking solution (0.3 or 0.1% Triton X-100, respectively, in PBS, 3 or 10% normal donkey serum and normal goat serum, respectively) and then incubated with the antibody of interests (Table I). The total number of cells in each field was determined by counterstaining cell nuclei with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich; 50 mg/ml in PBS for 15 min at RT). Immunostained sections and cells were mounted in Aqua-Poly/Mount (Polysciences) and analyzed at epi-fluorescent or confocal microscopy, using a Nikon Eclipse 90i microscope (Nikon) or a TCS SP5 microscope (Leica Microsystem). Z-stacks images were captured at 1 μm intervals with a ×40 or ×63 objectives and a pinhole of 1.0 Airy unit. Analyses were performed in sequential scanning mode to rule out cross-bleeding between channels. Fluorescence intensity quantification was performed with ImageJ software.