Pe anti ly 6g 1a8

The following antibodies were used for staining: BV510 or FITC anti-CD45 (30-F11), APC-Cy7 anti-CD3 (145–2C11), PerCP-Cy5.5 anti-NK1.1 (PK136), APC or PE-Cy7 anti-DNAM-1 (10E5), PE anti-F4/80 (BM8), APC anti-CD11b (M1/70), PE anti-NCR1 (29A1.4), PE anti-Ly-6G (1A8), PE anti-c-Kit (2B8), APC anti-CD49b (DX5), PE-Cy7 anti-CD200R3 (Ba13), PE anti-ICAM-1 (YN1/1.7.4), Alexa 488 anti-ICAM-2 (3C4), PE-anti PVR/Necl5 (TX56) (all from BioLegend, San Diego, CA), recombinant mouse DNAM-1 Fc chimera protein (R&D Systems), and Alexa Fluor 647 goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (Thermo Fisher Scientific). The following antibodies were used for in vivo blocking experiments: anti-LFA-1α (M17/4), anti-Mac-1 (M1/70) (both from Bio X Cell, West Lebanon, NH), and Rat IgG2a isotype control antibody (RTK2758; BioLegend). The following antibodies or reagents were used for in vivo cell depletion: anti-asialo GM1 (FujiFilm Wako Pure Chemical Corporation, Osaka, Japan), Rabbit IgG isotype control (Thermo Fisher Scientific), anti-Ly6G (1A8; BioLegend), Rat IgG2a isotype control, anti-CD200R3 (Ba103; Hycult Biotech, Uden, the Netherlands), Rat IgG2b isotype control (RTK4530; BioLegend), clodronate liposomes (Hygieia Bioscience, Osaka, Japan), or control liposome (Hygieia Bioscience).

Paraffin embedded sections were visualized using FITC channel only by the Zeiss LSM800 confocal microscope. For evaluating phagocytosis, SF samples were initially collected as described above. SF cells were filtered and stained with PE-anti-Ly-6G (1A8, BioLegend) plus Hoechst (33342, Invitrogen, ThermoFisher Scientific) for 30 min at ambient temperature. After the staining, cells were fixed, permeabilized (BD Cytofix/CytoPerm solution, described above), and further stained intracellularly with AF488-conjugated goat anti-mouse IgG2b (polyclonal, ThermoFisher Scientific) in 1X Perm solution for 1 h. After sufficient wash by the same Perm solution, cells were finally visualized under the 100X objective of the Zeiss LSM800 system using AF488, PE, and Hoechst channels, with compensations been optimized.

Paraffin embedded sections were visualized using FITC channel only by the Zeiss LSM800 confocal microscope. For evaluating phagocytosis, SF samples were initially collected as described above. SF cells were filtered and stained with PE-anti-Ly-6G (1A8, BioLegend) plus Hoechst (33342, Invitrogen, ThermoFisher Scientific) for 30 min at ambient temperature. After the staining, cells were fixed, permeabilized (BD Cytofix/CytoPerm solution, described above), and further stained intracellularly with AF488-conjugated goat anti-mouse IgG2b (polyclonal, ThermoFisher Scientific) in 1X Perm solution for 1 h. After sufficient wash by the same Perm solution, cells were finally visualized under the 100X objective of the Zeiss LSM800 system using AF488, PE, and Hoechst channels, with compensations been optimized.