Bovine serum albumin (bsa)

Mice were injected with pimonidazole hydrochloride in PBS (80mg/kg; Hypoxyprobe) intraperitoneally 1.5 hours prior to sacrifice. Tissues were dissected and processed as described above. For flow cytometry studies, pimonidazole was visualized using anti-pimonidazole antibodies (Pacific Blue Mab-1 clone 4.3.11.3; Hypoxyprobe) after cells were fixed for 20 min at 4°C using the FOXP3 Fix/Perm kit (BD Biosciences), and washed in permeabilization buffer. For imaging studies, dissected tumors were embedded in OCT and sectioned into 10μm cryosections. Cryosections were stored at −80°C until further use. For immunostaining, sections were fixed in 4% PFA (Electron Microscopy Sciences) for 20 minutes at RT, followed by a rinse in PBS containing 1% BSA (Sigma). Sections were blocked in 1% BSA in PBS containing anti-CD16/32 (BioXCell) for 1 hour at RT, and washed. Sections were stained with CD11b-AF594 (clone M1/70;BioLegend), CD31-AF647 (clone 390;BioLegend) and Pacific Blue Mab-1 (clone 4.3.11.3;Hypoxyprobe) for 1 hour at RT, followed by a wash and mounted using Vectashield (Vector Laboratories) and sealed with nail polish. Images were acquired on a Leica SP8 confocal microscope. Data analysis was performed using Imaris (Bitplane).