F4 80 biotin

PBS-perfused brains were immersion-fixed in 4% PFA for 24 h, then sucrose protected (20%) for at least 24 h and then embedded in OCT cryomount (Histolab) and frozen in isopentane. Sections were stained using the following antibodies: Iba-1 (Wako), F4/80-biotin (Cl:A3-1, AbD Serotec), GFP (Abcam, ab13970), GFAP (Abcam, ab7260), Ki67 (Abcam, ab15580), P2ry12 (generated by Dr. O. Butovsky), ICAM-1 (Abcam, ab119871). P2ry12-staining and CX3CR1⁺/Ki67⁺ cell counting was performed Pannoramic viewer and Histoquant software (3D histech).

Formalin-fixed paraffin-embedded tumor sections (5 μm) were immunolabeled for S100B to identify melanoma cells, and for Ki67 to identify proliferating cells. Sections were dewaxed in xylene and heat-treated in Target Retrieval Solution (DAKO). Non-specific antibody binding was blocked by incubating sections with 3% (v/v) hydrogen peroxide, biotin and avidin block (DAKO) and 10% (v/v) normal goat serum (DAKO). Rabbit anti-S100B (DAKO; 1:4,000) and rat anti-Ki67 (DAKO; 1:40) antibodies were then applied and incubated overnight at 4°C. S100B labeling was revealed with anti-rabbit HRP (DAKO) and AEC peroxidase substrate (Vector Laboratories), while Ki67 labeling was revealed with biotinylated donkey anti-rat (Jackson Lab; 1:300) followed by alkaline phosphatase-conjugated streptavidin (Rockland Inc.; 1:2000) and Alkaline Phosphate Substrate Kit III (Vector Laboratories). To visualize immune cells within the tumor bed we use anti-S100B (DAKO), CD45-APC (Biolegend; 1:200) and F4/80-biotin (AbDserotec; 1:50) antibodies overnight at 4°C. Anti-rabbit-FITC (DAKO; 1:500) and streptavidin-PE (Invitrogen; 1:500) antibodies were used to reveal S100B and F4/80 labeling respectively.