Rat anti cd68

Serial sections (1:24) were antigen retrieved (10 mM sodium citrate, 80 °C, 30 min), washed (1X phosphate buffered saline), blocked (5% Donkey Serum with 0.3% Triton-X), and incubated for 3 days at 4 °C with primary antibodies. Primary antibodies include rabbit anti-IBA1 (Wako, cat: 016-20001; 1:500), goat anti-IBA1 (Abcam, cat: ab107159; 1:1500), goat anti-GFAP (Santacruz Biotech, cat: sc-6170; 1:250), goat anti-GFAP (Novus Biological, cat: NB100-53809; 1:2000), rabbit anti-TGFβ1 (Abcam, cat: ab215715; 1:250), rat anti-CD68 (Biolegend, cat: 137002, 1:400), goat anti-Nestin (Novus biologicals, cat: NB100-1604, 1:400), rabbit anti-S100β (Abcam, cat: ab41548; 1:1500) and mouse anti-amyloid beta (6F3D) (Dako, cat: M0872; 1:200). After washing, antibodies were incubated in secondary antibodies (Jackson Immunoresearch; 1:200) for 1 h at room temperature, washed, and mounted. For plaque staining, sections were pre-treated for 5 min in 10% formic acid, followed by 10 min in 0.1 M borate buffer (PH 8.0). After 3 days of primary antibody, sections were incubated with anti-mouse biotin (Jackson ImmunoResearch, cat: 715-065-150; 1:200) for 2 h at room temperature, washed, and incubated with streptavidin-Alexa488 (Jackson ImmunoResearch, cat: 016-540-084; 1:400) for 1 h at room temperature, washed, and mounted.

Mice were deeply anesthetized by i.p. injection of 1% pentobarbital sodium and then transcardially perfused with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). The brains were removed and postfixed in 4% PFA overnight and then dehydrated in 30% sucrose for 72 h at 4 °C. Brains were then sliced into 30 μm coronal sections and mounted on slides. Slices were permeabilized and blocked with PBS containing 0.3% Triton X-100 and 10% NDS for 1 h at room temperature. Primary antibodies were incubated in PBS containing 0.3% Triton X-100 and 5% NDS overnight [rabbit anti-Iba1, 1:500 (WAKO, cat#019-19741); goat anti-Iba1, 1:1000 (Abcam, ab5076); rabbit anti-HMGB1, 1:100 (Abcam, ab18256); rat anti-CD68, 1:500 (BioLegend, cat#137001); mouse anti-vglut1, 1:500 (SYSY, 135011) and rabbit anti-c-Fos, (CST, cat#2250)]. The brain sections were then washed with PBS and treated with appropriate secondary antibodies conjugated with Alexa Fluor fluorophore in PBS containing 0.3% Triton X-100 and 5% NDS for 1 h at room temperature. The sections were rewashed with PBS and counterstained with DAPI (1:1000; Beyotime Biotechnology) for nuclear staining. Slides were coverslipped with 50% glycerin. All images were acquired with Leica TCS SP5 and Zeiss LSM880 confocal microscopes.