Mouse anti cd163

Cells were lysed in a buffer containing 50 mM Tris‐HCl (pH 7.4), 125 mM NaCl, 0.1% Triton X‐100, and 5 mM ethylenediaminetetraacetic acid with a 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA). We loaded 36 μg of sample in total volume of 20 μl. The resultant lysates were separated on 5–20% SDS‐polyacrylamide gels, transferred to membranes with iBlot Gel Transfer Stack (Invitrogen), and reacted with mouse anti‐CD163 (1:100, #10D6, Novocastra), mouse anti‐CD204 (1:500, #SRA‐E5, TransGenic), goat anti‐CD206 (1:100, #C‐20, Santa Cruz), and rabbit anti‐GAPDH (1:200, #FL‐335, Santa Cruz). After washing, the blots were incubated with HRP‐conjugated donkey anti‐mouse antibodies (1:1000, #NA934OV, Amersham, UK). The blots were then probed with ImmunoStar Reagents (Wako, Osaka, Japan).

After deparaffinization/hydration of sections, the sections were washed three times in TBST for 5 min each. The slides were boiled in 10 mM sodium citrate buffer, pH 6.0 and maintained at 121 °C for 10 min. The slides were cooled on the bench top for 30 min and washed in TBST three times for 5 min each. The sections were incubated in 3% H₂O₂ for 30 min and then washed in TBST three times for 5 min each. Sections were blocked with 100–400 µl blocking solution for 30 min at room temperature, followed by incubation with primary antibody overnight at 4 °C. We used mouse anti-CD163 (Clone 10D6; Novocastra, Newcastle, UK, 1:400 dilution), mouse anti-CD204 (Clone SRA-E5; Transgenic, Kumamoto, Japan, 1:200 dilution), rabbit anti-CD25 (Clone ab128955; Abcam, Cambridge, UK 1:200 dilution), rabbit anti-IL-10 (Clone ab34843; Abcam, 1:50 dilution), rabbit anti-PD-L1 (E1L3N; Cell Signaling Technology, USA, 1:200 dilution), and goat anti-CD69 (clone H-20; SANTA CRUZ, Heidelberg, Germany 1:50 dilution). Antibody was removed and 100–400 µl DAB (Peroxidase Stain DAB Kit®, Nacalai Tesque, Japan) was added to each section. We performed counterstaining with hematoxylin and washed the sections in dH₂O two times for 5 min each. After dehydration, we mounted the sections with coverslips.