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Immunohistochemistry stain was performed using a polyclonal commercial antibody (HPA017254 (Sigma-Aldrich)). After classic deparaffinization and rehydration, citrate buffer was used for antigen retrieval. Previously to incubation with secondary antibody, primary antibody was used to cover the samples for two hours. Chromogenic detection was performed with streptavidin-horseradish peroxidase conjugate (Universal LSAB kit, Dako Corp) and diaminobenzidine tetra-hydrochloride (liquid DAB, Dako Corp). Distilled water and hematoxylin (CatHE-M, Biocare Medical, CA, USA) were used for counterstaining. Finally, slides were mounted in Entellan medium (Merck & Co., NJ, USA). The entire procedure was performed by a highly experienced molecular biologist and expert pathologist.

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.

Swiss-rolled colons were fixed in 4% paraformaldehyde in sterile PBS for 24 h, transferred to 70% ethanol, and stored at 4°C. Paraffin embedding, sectioning, H&E staining, and Ki67 immunohistochemistry were performed at University of Florida Molecular Pathology Core. Briefly, 4-µm sections were deparaffinized and treated by Trilogy (REF:920P-06; CELL MARQUE) in a 95°C water bath for 25 min. Background Sniper (#BS966M; Biocare Medical) was applied for 15 min to reduce background. Sections were incubated with rat anti-mouse Ki67 (#M7249; 1:50, DAKO) for 60 min, followed by NB Rabbit anti Rat 1:200 for 30 min. The stain was visualized using Mach 2 Rabbit HRP polymer (# RHRP520L; Biocare Medical) and the 3,3'-diaminobenzidine) chromogen (#SK-4105; Vector Laboratories,) with modified Lillie-Mayer CAT hematoxylin counterstain (#CATHE-M; Biocare Medical). Slides were scanned using Keyence BZ-X800 microscope and VHX series software.