Immunohistochemical Staining of Brain Slices

Brain slices (30 μm) were prepared as previously described [19 (link)]. Sections were mounted on poly-D-lysine coated slides (Thermo Fisher), antigen retrieval with sodium citrate buffer was performed, except when staining with the TOC1 antibody, and the slides were blocked with PBS containing 5% BSA and 0.1% tween 20 for 1 h at room temperature. The sections were incubated with primary antibody in 5% BSA in PBS overnight at 4°C. The next day, slices were incubated for 1 h at room temperature with Alexa Fluor^™-conjugated secondary antibody including Alexa Fluor^™ 594 donkey-anti-rabbit, Alexa Fluor^™ 488 donkey-anti-rabbit, or Alexa Fluor^™ 647 donkey-anti-rabbit (Thermo Fisher Scientific). Alternatively, they were labeled using the MOM kit (BMK-2202, Vector laboratories), followed by three washes with PBS and labeling with Streptavidin Alexa Fluor^™ 488 or 647 (Thermo Fisher Scientific). The brain sections were coverslipped with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961). The slides were imaged using a Nikon A1R HD laser scanning confocal microscope and recorded by NIS-Elements (Version 5.11) software. Resulting images were pseudocolored for illustration purposes.