Proper secondary antibodies

Immunofluorescence staining was performed as previously described by our laboratory (Lu et al., 2017b (link)). In brief, coronal brain sections (25 μm) were incubated with buffer containing 10% normal donkey serum and 0.1% Triton X-100 for 1 h at room temperature followed by incubation with the corresponding primary antibodies in the same buffer overnight at 4 ° C. The following primary antibodies were used: anti-BrdU, Ki67 (DSHB); anti-NeuN, MAP2, DCX (Santa Cruz); anti-synaptophysin, spinophilin (Abcam); anti-Iba1 and GFAP (Proteintech Group). After incubation with primary antibodies, the brain sections were washed three times at room temperature followed by incubation with proper secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Sections were then washed with PBS, mounted with water-based mounting medium, and coverslipped in Vectashield mounting medium with DAPI (Vector Laboratories). LSM510 Meta confocal laser microscope (Carl Zeiss) was used to capture the fluorescence images. The imaging areas were chosen from the peri-infarct penumbral region beneath the infarct surface for comparison of immunofluorescent intensity between groups. For quantitative analyses, the number of NeuN-positive cells per 150 μm length in the peri-infarct zone was counted in 4–5 areas selected on each slice. 3–4 slices (50 μm apart) were chosen from each rat.