Ag 20b 0033

Tumor tissues were embedded in OCT (Sakura) by liquid nitrogen flash freezing. Tissue sections (4 μm thick) were baked at 37°C for 1 hour, fixed with ice acetone at -20°C for 20 minutes and washed with PBS. For cell immunofluorescence staining, cells on 20-mm diameter glass coverslips (NEST) were fixed with 4% paraformaldehyde at room temperature for 30 minutes and permeabilized with 0.4% Triton X-100 for 15 minutes. Slides or coverslips were blocked with 3% BSA and then incubated with the primary antibodies overnight at 4°C. After washing, slides or coverslips were incubated with secondary antibodies at room temperature for 1 hour and then incubated with DAPI for 30 minutes. Finally, slides or coverslips were washed with PBS, mounted and then imaged with confocal microscope (Zeiss LSM 780). The primary antibodies against: POSTN (Adipogen, AG-20B-0033, 1:400), α-SMA (Cell Signaling Technology, 19245, 1:200), EpCAM (Abcam, ab213500, 1:200), CD31 (Abcam, ab76533, 1:200), F4/80 (Abcam, ab300421, 1:200), Ki67 (Abcam, ab15580, 1:200) were used.

Tissue samples fixed in 4% paraformaldehyde were embedded in paraffin and sliced into 4 μm sections. The slides were baked for 2 hours at 65°C and then deparaffinized. Antigen heat retrieval was performed with pH6.0 citric buffer or pH9.0 EDTA buffer. Tissue sections were then stained with primary antibodies detailed in Table S1. Tissues were mounted using UltraSensitive^TM SP (Mouse/Rabbit) IHC Kit (KIT-9710) and imaged with Leica DM4B upright microscope. Antibodies against POSTN (Adipoge, AG-20B-0033, 1:400), α-SMA (Cell Signaling Technology, 19245, 1:200), EpCAM (Abcam, ab213500, 1:200), Ki67 (Abcam, ab15580, 1:200) and IL-4 (Abcam, ab300138, 1:200) were used.