GFAP with an accompanying Nissl stain was used for astrocyte quantification (Riquier & Sollars, 2020 (link)). Following PBS rinses (5 × 5 min 0.2% Triton X‐100), sections were blocked in 5% NGS for 90 min, incubated with rabbit anti‐GFAP antibody (1:13,000, Abcam, Cat. # ab7260, RRID: AB_305808), and then rinsed and blocked for 30 min in methanol containing 0.3% H2O2.
Following three rinses, both Iba1 and GFAP sections were placed in a goat anti‐rabbit antibody solution containing 2% NGS and 0.3% Triton X‐100 for 2 h at room temperature (1:1000, Vector Laboratories, Cat. # BA‐1000, RRID: AB_2313606). Sections were rinsed and then visualized using a 2% avidin–biotin complex solution containing 0.1% Triton X‐100 (Vector Laboratories, Burlingame, CA) for 1 h followed by a 0.05% diaminobenzidine solution containing 0.125% nickel ammonium sulfate and 0.01% H2O2. Every immunohistochemistry run contained negative control sections that were processed identically to experimental tissue except for omission of the primary or secondary antibody. No staining was observed on negative control tissue.
Following three rinses, both Iba1 and GFAP sections were placed in a goat anti‐rabbit antibody solution containing 2% NGS and 0.3% Triton X‐100 for 2 h at room temperature (1:1000, Vector Laboratories, Cat. # BA‐1000, RRID: AB_2313606). Sections were rinsed and then visualized using a 2% avidin–biotin complex solution containing 0.1% Triton X‐100 (Vector Laboratories, Burlingame, CA) for 1 h followed by a 0.05% diaminobenzidine solution containing 0.125% nickel ammonium sulfate and 0.01% H2O2. Every immunohistochemistry run contained negative control sections that were processed identically to experimental tissue except for omission of the primary or secondary antibody. No staining was observed on negative control tissue.
