Dylight 594 lectin

Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute medium (RPMI), fetal bovine serum (FBS), penicillin–streptomycin (P/S), 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), NucBlue fixed cell stained, goat anti-rabbit Alexa Fluor 488, TRIzol, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagents were purchased from Invitrogen (Carlsbad, CA). Lysine-coated glass-bottom dishes were purchased from MatTek Inc. (Ashland, MA). Anti-AIF antibody was purchased from Abcam (Cambridge, U.K.). Methanol, 4% formalin solution, Triton-X, bovine serum albumin (BSA), and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO). Mitochondria isolation for the mammalian cell kit and normal goat serum (NGS) were purchased from ThermoFisher (Waltham, MA). Lectin Dylight 594 was purchased from Vector Laboratories Inc. (Burlingame, CA). Phosphate-buffered saline (PBS) and Tris-buffered saline (TBS) were obtained from Corning Inc. (Corning, NY).

Rabbit kits were anesthetized and intracardially perfused with saline followed by 10% formalin at G29, PND1 and PND5. Brains were post-fixed for 24 h and cryoprotected in 30% sucrose. 30 μm sections were cut with a cryostat and mounted onto poly-L-lysine coated slides (Sigma Aldrich, MO, USA). The sections were blocked with 5% donkey serum, incubated with goat anti-IBA1 (1:250, Abcam, MA, USA), rat anti-CD11b (1:100, Genetex, CA, USA) and mouse anti-CD45 (1:100, Bio-Rad, CA, USA), or goat anti-IBA1 (1:250, Abcam, MA, USA) and rabbit anti-iNOS (1:50, Thermo Fisher Scientific, MA, USA), followed by fluorescent secondary antibodies (1:250, Thermo Fisher Scientific, MA, USA) for 2h. For TSPO and Lectin co-staining, sections were blocked with 5% donkey serum, and incubated with goat anti-TSPO (1:500, GeneTex, CA, USA) overnight at 4°C. The sections were washed, incubated with DyLight 594 lectin (1:250, Vector Laboratory) and fluorescent secondary antibody (1:250, Thermo Fisher Scientific, MA, USA) for 2 h. Sections were washed and incubated in DAPI (1:1000) for 15 min. Immunolabelled sections were cover-slipped with Dako fluorescence mounting medium (VWR, PA, USA). All the images were captured on Zeiss LSM710 confocal microscope, keeping similar settings during image acquisition for different groups.