Immunofluorescence Staining of Mouse Brain

Immunofluorescence (IF) staining of mouse brain sections was performed as previously described (31 (link)). Slides were washed 3 times for 15min with PBS to remove residual OCT. The sections were then incubated in the blocking solution (PBS containing 10% donkey serum (cat no: S30-100ml, Millipore Sigma), 2% BSA (Fisher Scientific, BP1600-100) and 0.3% Triton X-100 (Fisher Scientific, BP151-100) for 2h at room temperature (RT). Sections were then transferred to blocking solution containing the primary antibodies (NBR1 (proteintech, 16004-1-AP), IBA1 (Novus Biologicals, NB100-1028), and incubated overnight at 4°C. After that, sections were washed with PBS 3 times for 15min each. Then, they were incubated with the blocking solution containing the secondary antibody Donkey anti-Rabbit IgG (ThermoFisher Scientific, A32790), Donkey anti-Goat IgG (cat. no: A-11058, ThermoFisher Scientific) for 2h at RT. DAPI (Fisher Scientific, D1306) was added on the top of the antibody solution in the last 15min of the incubation period at a final concentration (5ug/ml). Finally, sections were washed with PBS 3 times for 15min. Antifade mounting media (ThermoFisher Scientific, P36934) was added before cover-slipped.