Rat anti f4 80 antibody

The following primary antibodies were used: rabbit anti-RANKL antibody and rabbit anti-OPG antibody (Bioss Antibodies, Boston, MA, USA), sheep anti-CSF-1R antibody (R & D systems, Minneapolis, MN, USA), rabbit anti-cathepsin K antibody (Abcam, Cambridge, United Kingdom), rat anti-CSF-1R antibody coupled to phycoerythrin (PE) (AFS98), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F 11), and rat anti-Ter119 (TER-119) antibody coupled to APC (Thermo Fisher Scientific, Waltham, MA, USA), rat anti-F4/80 antibody coupled to FITC (BM8) and rat anti-F4/80 antibody coupled to PE (BM8) (BioLegend, San Diego, CA, USA), rabbit anti-HIF-1α antibody (GeneTex, Irvine, CA, USA), and rabbit anti-HIF-2α antibody (Novus Biologicals, Centennial, CO, USA).
The following secondary antibodies were used: donkey anti-sheep coupled to Alexa Fluor (AF) 488 (Thermo Fisher Scientific) and goat anti-rabbit coupled to AF Plus 555 (BD Biosciences, San Jose, CA, USA). Nuclei were stained using Hoechst 33342 (Thermo Fisher Scientific) or Propidium iodide (PI) (BD Biosciences).

Subcutaneous mouse tumors were dissected, weighed, and then placed in six-well plates with growth medium (RPMI, 5% FBS) and minced. Minced tissues were combined into three groups per genotype, spun down in 50 mL conical tubes, and resuspended in 20 mL digestion buffer (0.5 mg/mL collagenase IV, 0.1 mg/mL DNase I in HBSS medium). Tumors were digested at 37°C with gentle shaking for 30 min and vortexed every 10 min. Contents were filtered through a 100 mm mesh, and cells were pelleted and resuspended in 45% percoll in 1X HBSS and 1X PBS. Cells were spun at 2000 rpm at 4°C with a swing bucket rotor for 20 min. The supernatant was aspirated, and the pellet was briefly resuspended in 5 mL ACK buffer to lyse red blood cells. Last, cells were pelleted and resuspended in growth medium for downstream applications.
To isolate F4/80⁺ cells from tumors, tissues were homogenized and processed as above to collect tumor-infiltrating leukocytes. F4/80⁺ cells were then isolated by positive selection using a rat anti-F4/80 antibody (Biolegend, 123120) and sheep anti-rat Dynabeads (ThermoFisher Scientific, 11035) according to the manufacturer’s instructions.