Goat anti rabbit alexafluor647

Tumors were cut in half and embedded in optimal cutting temperature compound (OCT) and frozen on dry ice before storing at −80 ˚C. Frozen tissues were the sectioned (5 microns thick) using a cryostat. Tissue slides were fixed in cold methanol in −20 ˚C for 10 min, washed with PBS +0.01% Triton X-100, then pure PBS, blocked with 5% goat serum at room temperature for 1 hour, and stained with purified primary antibody at 4˚C overnight. Tissues were then washed with PBS three times, stained with secondary antibodies at room temperature for one hour, counterstained with Hoechst Dye and mounted in Prolong mounting media (Thermo Fisher Scientific). The following antibodies were used for detecting mouse antigens by immunofluorescent staining: CD4 (eBioscience, clone RM4–5), CD8 (eBioscience, clone 53–6.7), K14 (eBioscience, polyclonal Cat#PA5–16722), K17 (provided by Pierre A Coulombe [57]), Goat anti-rabbit AlexaFluor647 (Molecular Probes), Goat anti-rat AlexaFluor 488 (Molecular Probes).