Clone 4sm15

Harvested subcutaneous tumors were formalin-fixed and paraffin-embedded. Sectioned tissues were then deparaffinized and soaked in 0.3% H₂O₂ in methanol at RT for 10 minutes to extinguish endogenous peroxidase activity. Antigen retrieval was performed by heating specimens in a sodium citrate buffer solution using a microwave. After cooling, sections were incubated in Peroxidase Blocking Reagent (Dako, Santa Clara, USA) for 10 minutes at RT. Sectioned tissues were incubated with primary antibody against CD8 (clone 4SM15, 1:100 dilution, eBioscience, San Diego, USA) or FoxP3 (clone FJK-16s, 1:100 dilution, eBioscience) or aSMA (A5228, 1:1,000 dilution, Sigma-Aldrich) or CD4 (clone 4SM95, 1:100 dilution, eBioscience) or TGF-β (ab215715, 1:100 dilution, Abcam, Cambridge, UK) for 60 minutes at RT. Following three 5-minute washes with PBS, sections were incubated with secondary antibody for 30 minutes at RT. After washing, the enzyme substrate 3,30-diaminobenzidine (Dako, Santa Clara, USA) was used for visualization, and sections were counterstained with Meyer’s hematoxylin. Evaluation of sections were performed using ImageJ software. The number of cells expressing CD8, FoxP3, CD4 were counted in four randomly selected high-magnification fields. The scores of αSMA and TGF-β were evaluated using an “area index,” calculated in low magnification fields.

Forty-μm thick free-floating sections were washed in 0.01 M tris-buffered solution (TBS; pH 7.4) 3 X for 5 min. The tissue then underwent a sodium citrate antigen retrieval (0.1M; pH 7.3) process for 30 min at 37 °C. After antigen retrieval the free-floating sections were washed and blocked in 5% normal serum for 1–2 h. The sections were thereafter incubated in 1% serum in TBS-Triton (TBST) primary antibody solution consisting of one of the following antibodies: anti-Iba1 (1:500, WACO), anti-Olig2 (1:250, clone SP07-02; R&D), anti-GFAP (1:500, clone DIF48; Abcam), anti-CD3 (1:500, clone 17A2; Thermo Fisher), anti-CD8 (1:500, clone 4SM15; eBioscience), anti-Thy1.1 (1:500, OX-7; Invitrogen) anti-CD4 (1:500, clone RM4-5; Thermo Fisher), anti-NK1.1 (1:250, clone EPR22990-12; Abcam). After an overnight incubation, the free-floating sections were washed and put into a 1% serum TBST secondary solution for 2 h. Sections were mounted onto coated glass slides, and cover slipped using hard set mounting medium (Vector Laboratories). Fluorescence images were collected on a Ti2 Nikon microscope using a Ci2 confocal system.