Immunofluorescence Staining of Mouse Tumor Sections

Immunofluorescence was performed as previously described^{13 (link),14 (link)}. In brief, mouse tumor sections were de-paraffinized and rehydrated, then subjected to antigen retrieval in Target Retrieve Solution (DAKO; S1699) at 95°C for 20 min. Sections were blocked with 5% horse serum for 1 h at room temperature, incubated with anti-CD31 (1:100; Dianova; DIA-310; for mouse tissues), anti-FSP-1 (1:100; Millipore; 07–2274), anti-tdTomato (1:100; Rockland; 600-401-379) or anti-VCAM-1 (1:100; Cell Signaling Technology; 12367) antibody overnight at 4°C. For cell culture staining, the cells were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 1% Triton X-100 for 5 min. Cells were blocked with 5% horse serum for 1 h, then incubated with Alexa Fluor 488-conjugated phalloidin (1:100; Invitrogen; A12379) for 20 min. Images were acquired using an Axio Imager microscope (Zeiss) equipped with an AxioCam 506 monochrome CCD camera (Zeiss).