Immunofluorescence Analysis of Brain Sections

Immunofluorescence analysis was performed on sections (7 μm) of brain as previously described [72 (link)]. Briefly, after deparaffinization, the sections were boiled in 0.1 M citrate buffer for 1 min. Non-specific adsorption was minimalized by incubating in 2% (v/v) standard goat serum in PBS for 20 min. Slides were incubated overnight with anti-BrdU (1:100, SCB, #sc-32323) antibodies. Sections were washed with PBS and were incubated with secondary antibody fluorescein (FITC)-conjugated anti-mouse Alexa Fluor-488 antibody (1:2000 v/v, Molecular Probes, UK) for 1 h at 37 °C. Images were collected using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) following a typical procedure [62 (link)]. Each picture was digitalized and analyzed as previously described [54 (link),73 (link)].