Anti cd86

Cell slides were de-paraffinized, rehydrated with an ethanol gradient, and 50% TritonX was added dropwise to the tissue and left for 10 min at room temperature. They were then washed with PBS thrice with 5 min each time. Citrate buffer was added to the slide and antigen retrieval was carried out at high temperature for 5 min. The slides were washed with 0.01 M PBS and closed with goat serum for l0 min at room temperature. Sections were incubated overnight at 4 °C with primary antibodies anti-CD86 (1:50; BIOSS, Beijing, China), anti-IL-6 (1:50; BIOSS, Beijing, China), anti-CD206 (1:50; Abcam, Shanghai, China), anti- IL-10 (1:50; Abcam, Shanghai, China). Unbound primary antibodies were washed off with PBS, and sections were incubated with secondary antibodies followed by nuclear staining and image acquisition. Lung tissue sections were incubated with primary antibodies anti-CD80 (1:50; BOSTER, Wuhan, Hubei, China), anti-IL-1β (1:100; BOSTER, Wuhan, Hubei, China), anti-IL-10 (1:50; BOSTER, Wuhan, Hubei, China) and anti-F4/80 (1:100; BOSTER, Wuhan, Hubei, China). Subsequent steps were the same for cellular immunofluorescence.

Tumor or brain tissues were harvested, fixed in paraformaldehyde, embedded within paraffin and sectioned. Tumor sections (10 μm) were deparaffinized and rehydrated. Sections were boiled in 10 mM citrate buffer (pH 6.0) at 95 °C for 15 min for antigen retrieval. Sections then were blocked with 5% goat serum in PBS for 1 h and incubated with anti-CD8 (1: 800) (Servicebio, China), anti-CD49b (1: 400) (Bioss, China), anti-CD86 (1: 500) (Bioss, China) and anti-TNF-α (1: 500) (Bioss, China) at 4 °C overnight, followed by incubation with Alex 488- or Cy5-conjugated goat-anti-rabbit IgG (1: 500) (Servicebio, China) at room temperature for 1 h. Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using LSCM.
Optical imaging data were analyzed using ImageJ Fiji software32. Total cell numbers were calculated from manual counts of DAPI-labeled nuclei; counts were performed with the Fiji cell counter tool.

Paraffin sections were deparaffinized and dehydrated. Next, the sections were blocked with 10% goat serum for 1 h at 37°C after antigen retrieval. The sections were incubated with primary antibodies, including anti-F4/80 (1:200; Bioss), anti-CD86 (1:200; Bioss) and anti-CD163 (1:200; Santa Cruz, USA), overnight at 4°C. After being washed three times, the sections were incubated with fluorescent-conjugated secondary antibodies (1:200; CST, USA) for 1 h at 37°C and then 4′,6-diamidino-2-phenylindole solution was used for nuclear staining. The sections were observed under a fluorescence microscope at 400× magnification (Nikon Corporation, Tokyo, Japan).