Brain Tissue Immunohistochemistry Protocol

GFAP and Iba1 staining was performed according to our previous report^{44 (link)}. The brains were removed, postfixed overnight in 4% buffered PFA at 4 °C after perfusion and cryoprotected in 30% sucrose. Brains were frozen in ethanol with dry ice, and 10-μm-thick sections were prepared using a Cryostat. The sections were blocked and permeabilized with PBS including 10% normal goat serum (Sigma-Aldrich) and 0.3% Triton-X 100 for 1 h at room temperature. The sections were incubated with primary antibody (Anti-GFAP, no dilution, Dako/Agilent, Santa Clara, CA, USA; Anti-Iba1, 1/500, Wako, Osaka, Japan) for 3 h at room temperature, followed by Alexa-labeled secondary antibody for 1 h at room temperature in the dark. The sections were mounted on a glass slide with DAPI-Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Images were obtained with an inverted fluorescence microscope (BZ-9000, Keyence, Osaka, Japan) and were processed using ImageJ software (NIH, MD, USA).