Citrate buffer

CD68+ macrophages were detected in mouse tissues by immunohistochemistry using a CD68 (FA-11) rat monoclonal antibody (ThermoFisher Scientific, Rockford, IL, USA) at a dilution of 1:200. After deparaffinization, epitope retrieval was performed by immersing the sections in citrate buffer (Scytek, UT, USA), followed by heating in a pressure cooker. Thereafter, sections were successively exposed to solutions containing avidin and biotin to avoid false-positive staining reactions resulting from endogenous biotin, followed by casein and anti-CD68 incubation overnight. The day after, sections were exposed to the corresponding biotinylated secondary antibody (polyclonal rabbit anti-rat IgG) and then to the avidin-biotin-peroxidase complex (ABC kit), both from Vectorlab (Peterborough, UK). Presence of antigen in the sections was visualized by incubation with a chromogenic substrate mixture containing DAB and H₂O₂. Finally, sections were counterstained with luxol fast blue and mounted with a synthetic medium. The number of CD68+ macrophages was counted in 5 fields in each tumor tissue with an AxioCam MRC5 optical microscope at 400× magnification.

Paraffin-embedded lung tissue sections first underwent antigen retrieval with citrate buffer (Scytek), followed by incubation with anti-IL10 antibody for 24 h. Next, slides were colored with AF488 conjugated donkey-anti-rat secondary antibody for 2 h. Slides were then incubated with mouse anti-human CD20 antibody for 2 h, followed by coloring with AF647 conjugated donkey-anti-mouse secondary antibody and DAPI. Appropriate isotype controls were used during the staining procedure to test for non-specific interactions. The antibodies used for immunohistochemistry can be found in Additional file 1: Table S2.

For the colon cancer tissue analysis, formalin-fixed, paraffin-embedded AccuMax tissue arrays were purchased from ISUABXIS with the approval of the Institutional Review Board in Hallym University. The colon cancer tissue array (A203VII) is composed of 45 cases of colon cancer tissues in duplicate and 8 normal colon tissues. The slides were deparaffinized with xylene and rehydrated in ethanol, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. For antigen retrieval, all sections were boiled in a citrate buffer (pH 6.0) (ScyTek Laboratories) for 15 min. The slides were incubated overnight in PBST (PBS, Tween 0.2%) containing the anti-TM4SF5 monoclonal antibody (10 μg/ml) at 4°C, followed by incubation with biotinylated anti-mouse IgG antibody (Histostain Plus kit, Invitrogen). They were sequentially reacted with streptavidin-conjugated peroxidase, 3',3'-diaminobenzinidine (0.5 mg/ml) and hydrogen peroxide and then counterstained with hematoxylin. After rinsing, the sections were mounted, dehydrated, and covered with cover slips. All images were examined using a Nikon Eclipse E-200 microscope. The percentages of cells expressing TM4SF5 were calculated as the number of TM4SF5-positive cells divided by the total number of cells in each tumor type.