Apc cd4 rm4 5

Fresh tumor tissues were fixed in 4 % paraformaldehyde solution for 48 hours, and the tissues were dehydrated and embedded in paraffin. Immunohistochemistry (IHC) analysis of the embedded tissue was performed in 5 mm thick sections. In brief, primary antibodies, 4-HNE (1:400, ab46545, Abcam) were incubated overnight at 4 C. Staining was performed using Rabbit polymer assay system (ZSGB-BIO) and DAB peroxidase substrate Kit (ZSGB-BIO). Images (5 per tumor) were randomly acquired from the slice using an Axio Zoom.V16 microscope (Agilent) at 400 3 magnification.
Tumor infiltration lymphocyte analysis 4T1 xenografts isolated from BALB/c mice were sliced and digested with collagenase/hyalurinidase (Stemcell Technologies, Vancouver, BC, Canada) and DNase (Sigma). Lymphocytes were enriched using HistopaqueÒ-1083 (Sigma) and then T cells were enriched using the Dynabeads untouched mouse T cell kit (Invitrogen). The isolated T cells were fixed with 4 % paraformaldehyde for 5min and stained with PE-CD3ε (145-2C11; Biolegend), PE-Cyanine7-IFN-g (XMG1.2; Biolegend), APC-CD4 (RM4-5; Biolegend) and FITC-CD8a (53-6.7; BD PharmingenTM) at room temperature for 15 min in the dark. After three times washed, the populations of infiltration T cells were detected and analyzed with BD FACS (LSRF Fottessa) cytometer.