Cd4 d7d2z

Omental tumor samples were analyzed using H&E stain and immunohistochemistry. Paraffin slide sections were washed three times in xylenes followed by rehydration in a series of two alcohol washes (100% and 95%). Sections were treated for heat antigen retrieval in EDTA solution (1 mM, pH 8.0). After blocked in goat serum, sections were incubated overnight with specific primary antibodies as follows: PCNA (PC10) for proliferating cells, F4/80 (D2S9R) for Mφ, Arginase-1 (D4E3M) for MDSCs, CD4 (D7D2Z) for CD4 T cells, and CD8α (D4W2Z) for CD8 T cells (Cell Signaling Tech., Danvers, MA, USA). Sections were incubated with secondary antibody matched with primary antibody and developed using SignalStain^® DAB Substrate kit (#8059; Cell Signaling Tech.) with hematoxylin counterstain. Digital images of slides were captured with BZ-X700 All-in-One Fluorescence Microscope (KEYENCE). Quantitative analysis of inflammatory area in H&E stain was calculated using ImageJ with selection of inflammatory area through color threshold followed by particles analysis. Quantitative analysis of PCNA and immune cell dispositions was performed using ImageJ with brown color selection through color deconvolution of hematoxylin and 3,3′-diaminobenzidine (DAB) staining followed by intensity analysis. Quantitative analysis was obtained from 3–5 random fields of each slide (10×).

Tissue was fixed in 10% formalin and processed in the Molecular Pathology Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University for H&E and IHC. IHC anti-CD19 (D4V4B), CD8a (D4W2Z), and CD4 (D7D2Z) antibodies were purchased from Cell Signaling Technology (CST), and F8/40 (BM8) from eBioscience was used at manufacturer’s recommended dilutions. Goat anti IgG H+L biotinylated (Vector Laboratories) antibody was used to detect rat IgG anti-mouse PD-1. Slides were scanned using Leica SCN400 and visualized with Aperio ImageScope. Two blinded pathologists assisted with grading of immune infiltration. Two slides were made from each organ. Five random high-power filed images were scored and averaged.