Clone afs98

Liver specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 μm thickness. Antigen retrieval was achieved with citrate buffer. Tissue sections were stained overnight at 4°C with anti–mouse F4/80 (1:100 dilution; clone BM8), Csf1r (1:200 dilution; clone AFS98; both BioLegend), or Ki-67 (1:200 dilution; clone 16A8; Vector Laboratories). After being washed with PBS, slides were incubated with a fluorochrome-conjugated secondary antibody and then analyzed and imaged on an Axio Imager 2 wide-field microscope (Zeiss).

To establish syngeneic tumor models, B16F10 (1 × 10⁶ cells to C57BL/6), CT26 (5 × 10⁵ cells to BALB/c), and EG7-OVA (1 × 10⁶ cells to C57BL/6) were injected subcutaneously to the right flank of mice. For lung metastasis, B16F10 (1 × 10⁵ cells) were injected intravenously to B16F10 tumor bearing mice, 4 days after subcutaneous injection. For dual flank model, 1 × 10⁶ and 5 × 10⁵ B16F10 cells were injected subcutaneously to the right and left flank of mice, respectively. When tumor reaches an average volume of 50–100 mm³, the mice were randomized into several groups according to the experimental protocol. Tumor volume (mm³) was calculated as (width)² × (length) × 0.5. IT dosing on the right tumors was performed three times with 3 days interval. To establish a mouse orthotopic hepatocarcinoma model, Hepa1-6 cells (1.5×10⁶) in HBSS medium were injected into spleen of C57BL/6 mice and the spleen was excised subsequently. IV dosing was performed three times with 3 days interval from Day 4 after cell inoculation. Isotype control antibody (10 mg/kg, BioLegend), Anti-CD8 (10 mg/kg, BioLegend, Clone 53–6.7), Anti-NK1.1 (10 mg/kg, BioLegend, Clone PK136), Anti-CSF1R (10 mg/kg, BioXcell, Clone AFS98), Anti-PD1 (10 mg/kg, BioLegend, Clone RMP1–14), and Anti-CTLA4 (5 mg/kg, BioLegend, Clone 9H10) were administered intraperitoneally.