C0563

Brains were removed and fixed in 4% paraformaldehyde in PBS after transcardial perfusion, sectioned at 50–80 μm on a vibratome (Leica VT1000S) and processed for immunohistochemistry as free-floating sections. Sections were blocked in 1×PBS / 5% normal goat serum / 0.3% Triton^™ X-100 buffer solution at room temperature for 1 hour. Sections were then incubated with the diluted primary antibody solution overnight at 4°C. Primary antibodies included Rabbit anti-Olig2 (1:500, Sigma, AB9610), chicken anti-GFP (1: 1,000, Aves GFP-1020), mouse anti-NeuN (1:250, Sigma, MAB377). Sections were then washed 3 times in 1×PBS for 5 minutes each and transferred to the diluted secondary antibody solution in the blocking buffer at room temperature for 2 hours. Fluorescently conjugated secondary antibodies were obtained from Molecular Probes (AlexaFluor 488, 594 or 647, 1:500). Sections were then washed 3 times in 1×PBS for 10 minutes each, and incubated with nuclei staining solution (Molecular Probes H1398, 1: 5,000). Sections were mounted with glycergel(Dako, C0563) for imaging.

Tissues were fixed with 4% formaldehyde for 24 h and embedded in paraffin. Five-mm-thick FFPE tissue sections on glass slides were baked at 37 °C overnight, deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Next, tissue sections were incubated in retrieval solution (pH 6 or 9) for antigen retrieval at 95 °C for 30 min. Tissue sections were incubated in 3% hydrogen peroxide and in serum-free protein block solution (Dako, X0909) before adding the primary antibody for 1 h at room temperature. After signal amplification using the EnVison+ System HRP Labeled Polymer anti-mouse (Dako, K4001) or anti-rabbit (Dako, K4003) antibodies, chromogenic revelation was performed using 3-amino-9-ethylcarbazole (Vector Laboratories, SK4200). Slides were counterstained with hematoxylin, mounted with a glycerol-based mounting medium (Dako, C0563) and scanned for digital imaging (Hamamatsu NanoZoomer S60 Whole Slide Scanner). Next, the same slides were successively stained as described previously^{37 (link)}. Primary antibodies used are listed in Table 2.
Images were analyzed (automatic analysis) using QuPath software (https://qupath.github.io/).

Tissues were fixed in 4% formaldehyde for 24 h and embedded in paraffin. Four-millimetre-thick formalin-fixed paraffin-embedded (FFPE) tissue sections on glass slides were baked at 37 °C overnight, deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Tissue sections were incubated in citrate buffer (pH 6) for antigen retrieval at 95 °C for 30 min. Tissue sections were incubated in 3% hydrogen peroxide and in serum-free protein block solution (Dako, X0909) before adding primary antibody for 1 h at room temperature. After signal amplification using secondary antibody conjugated to streptavidin-horseradish peroxidase and chromogenic revelation using 3-amino-9-ethylcarbazole (AEC) (Vector Laboratories, SK4200), slides were counterstained with haematoxylin, mounted with a glycerol-based mounting medium (Dako, C0563) and scanned for digital imaging (Leica Biosystems, Aperio AT2 Digital Whole Slide Scanner). B16 lung tumours were pretreated with Melanin Bleach kit (Polysciences, 24909) before immunohistochemistry staining. Then the same slides were successively destained and restained with subsequent antibodies as described previously^{46 (link)}. The primary antibodies used are included in Supplementary Table 7. ROIs and quantification of FOXP3⁺ T_reg cells, CD4⁺ and CD8⁺ T cells were manually selected using QuPath software^{47 (link)}.