Rat igg2a isotype control antibody

For hematoxylin and eosin staining, ear specimens were fixed with 4% paraformaldehyde/PBS solution, embedded in paraffin, cut into 5 μm-thick sections, and stained with hematoxylin and eosin (Muto pure chemicals). For immunohistochemical staining, ear sections were treated with microwaves, followed by incubation with methanol containing 0.3% H₂O₂, avidin/biotin blocking kit (Vector laboratories, catalog#: SP-2001), and normal serum (Vector laboratories). Ear sections were then incubated with the following antibodies at 4 °C for 18 h: rat anti-Ly6G antibody (1 μg/mL; clone: 1A8, catalog#: 127602; BioLegend), rat IgG2a isotype control antibody (1 μg/mL; clone: RTK2758, catalog#: 400502; BioLegend), rabbit polyclonal anti-RIPK1 antibody (2.5 μg/mL, catalog#: NBP1-77077, Novus Biologicals), and rabbit polyclonal IgG isotype control antibody (2.5 μg/mL, catalog#: ab37415, Abcam). They were incubated subsequently with biotinylated secondary antibody, ABC reagent (Vector laboratories), 3´-diaminobenzidene tetrahydrochloride solution (Nacalai tesque), and counterstained with hematoxylin. TUNEL staining was conducted by using TACS 2 TdT-DAB In Situ Apoptosis Detection Kit (R&D systems, catalog#: 4810-30-K), according to manufacturer’s protocols.

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).

(1) Construction of CD137L-expressing cell lines. TrueORF cDNA expression clones for C-terminal Myc- and DDK (Flag)–tagged M. musculus TNFSF9 were obtained (cat. # MR220933, Origen). Circular plasmid DNA was isolated and restricted with ApaLI (NEB) to linearize the vector DNA. Linearized DNA was then transfected into HEK293 cells using Lipofectamine 2000 and stable integrants selected for using 400–500 μg/ml of Geneticin (G418). After isolation and expansion of individual cell clones, high mouse CD137L-Myc-DDK expressers were stained with rat anti-mouse CD137L antibody (clone: TKS-1, BioLegend) or Rat IgG2a isotype control antibody (clone: RTK2758, BioLegend), followed by biotinylated mouse anti-Rat IgG2a antibody (clone: RG7/1.30, BD Biosciences) and streptavidin PE (BioLegend), identified using BD FACSCanto (BD Biosciences).
(2) Confirmation of specific binding of sCD137 to CD137L. HEK-mCD137L or control HEK293 cells (2 × 10⁶) were preincubated with 1 ug/sample of anti-CD137L Ab (TKS-1) or the same volume of PBS for 30 min at 4°C. After washing cells to remove excess CD137L antibody, cells were incubated with titrated doses of sCD137 for 30 min at 4°C. Then cells were stained with biotinylated rat anti-mouse CD137 antibodies (clone: 17B5, BioLegend) followed by streptavidin PE (BioLegend). The level of bound CD137 was analyzed using FACSCanto (BD Bioscience).