Cd11b m1 70

Immunofluorescence was performed on OCT-embedded frozen tissue sections. Sections were blocked with 10% goat serum and 10% BSA in PBS for 1 hour at RT, incubated with primary antibody overnight at 4°C, then washed and incubated with Al-448 or Al-466 goat anti-rat secondary antibody (Invitrogen) at 1∶800 dilution. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tumor sections. Sections were subject to heated antigen unmasking solution for 30 minutes, quenched in 0.3% hydrogen dioxide in PBS for 15 minutes, blocked with 10% goat serum and 10% BSA in PBS for 1 hour at RT, then incubated with primary antibody overnight at 4°C, incubated with biotinylated goat anti-rat secondary antibody at 1∶800 dilution for 30 minutes at RT, and developed using the ABC kit (Vector Labs). Slides were counterstained with hematoxylin. Sections were mounted with Vectorshield and analyzed by fluorescence or light microscopy (Zeiss). Antibodies include CD11b (M1/70, R&D Systems), CD4 (RM4–5, Biolegend), CD8 (53.6–7, R&D Systems), CD49b (DX-5, Biolegend), Foxp3 (FJK-16s, eBioscience), Ly6G (RB6-BC5, eBioscience), Ly6C (AL-21, BD Biosciences), E-Cadherin (MAB7481, R&D Systems), N-cadherin (H-63, Santa Cruz Biotechnology), and phospho-IκBα (Ser 32/36, #9246, Cell Signaling).