Primary anti gfap antibody

The embedded brains in OCT were cut into coronal sections (10 μm) using a Tissue-Tek cryostat and mounted onto charged glass slides. Prior to staining, slides were washed with PBS (3x, 5,5, and 10 min); then, they were treated with blocking buffer (5% normal goat serum, 0.2% Triton X-100, 0.5% bovine albumin in PBS) for 1h at room temperature. The treated sections were then incubated overnight at 4°C with primary anti-GFAP antibody (1:100 dilution, Biolegend San Diego, CA, USA) or anti-IBA1 antibody (1:250 dilution, FUJIFILM Wako Chemicals U.S.A Corporation Richmond, VA, USA). For OCTN1 staining, the slides were treated with OCTN1/2 (H-9) antibodies (1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). Then, the slides were washed with PBS (3x) for 10 min each, the sections stained for GFAP and OCTN1 were subsequently incubated with secondary antibody goat anti-mouse Alexa Fluor 488 (1:200 dilution, Thermo Fisher Scientific, Carlsbad, CA, USA). Sections stained for IBA1 were incubated with secondary antibody goat anti-rabbit Alexa Fluor 680 (1:200 dilution, Thermo Fisher Scientific, Carlsbad, CA, USA) for 30 min at room temperature. The sections were then washed with PBS twice for 10 minutes and once for 30 minutes, and cover slipped with an antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA) before observation under a fluorescence microscope.