Ab80437

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.