Expose rabbit specific hrp dab detection kit

MC38 tumors were embedded in optimal cutting temperature (OCT) compound, snap-frozen on dry ice, and stored at -80°C until staining. Serial 5 μm tumor sections were placed onto glass slides, dried overnight at room temperature (RT), and then stained or stored at -80°C until use. For immunohistochemical staining, air-dried slides were fixed with acetone at RT, treated with peroxidase block (Abcam) to quench endogenous peroxidase, and then further blocked with a 10% goat serum and 5% BSA solution. Sections were stained with rabbit anti-mouse CD3 antibody (Abcam SP7) at a 1:400 dilution or rat anti-mouse PD-L1 (eBioscience MIH5) at a 1:100 dilution in blocking buffer. CD3⁺ T cells were detected using the EXPOSE rabbit-specific HRP-DAB detection kit (Abcam ab80437). PD-L1⁺ cells were detected using goat anti-rat-HRP (1:1000; Jackson ImmunoResearch 112-036-071) and detected with the HRP-DAB detection kit. Slides were co-stained with pre-made hematoxylin (Biocare CATHE-M) to counterstain cell nuclei. A similar protocol was applied for staining of PD-L1.

Immunohistochemistry was performed to detect six immunomarkers (S3 Table). Slides were deparaffinized in xylol and rehydrated in a series of decreasing ethanol concentrations. Antigen recovery was performed for 45 min at 96°C in a citrate buffer (pH 6.0). Next, the slides were washed (TBS-T) thrice for 5 min each and incubated with 0.1% H₂O₂ ready for use solution (ab80437 Abcam, UK) for 10 min. Protein blocking solution was applied for 10 min, and after washing in TBS-T, the slides were incubated overnight at 4°C with primary antibodies (diluted in 1% albumin). Slides for detecting FOXP3, CD1a, CD4, and CD8 (Abcam, UK) were incubated with HRP polymer (ab210059, Abcam, UK) for 30 min at 25°C, and those for CD3 and IFN-γ (Abcam, UK were incubated with a secondary HRP-conjugated rabbit antibody (ab80437, Abcam, UK) for the same time (specific targets and dilutions of the primary antibodies are shown in S3 Table). For rabbit-raised antibodies, a biotin-free immunoenzymatic detection kit was used (Expose Rabbit Specific HRP/DAB Detection Kit, ab80437 Abcam, UK), whereas for mouse-raised antibodies, a double-stain IHC kit (M&R on human tissue, DAB & AP/Red, ab210059, Abcam, UK) was used. The slides were stained with the chromogenic substrate DAB (3,3’-diaminobenzidine tetrahydrochloride) (ab210059, Abcam, UK) and counterstained with hematoxylin.