Mouse anti human id1 antibody

RA, OA, and normal (NL) (no arthritis) ST cryosections as well as ankle sections of Wt mice induced with K/BxN serum were fixed in cold acetone for 30 min at 4 °C. The tissue sections were blocked with 5 % donkey serum and 20 % fetal bovine serum (FBS) in phosphate-buffered saline (PBS) at 37 °C for 1 h. The sections were then incubated with either mouse anti-human Id1 antibody (Abcam, Cambridge, MA, USA, 10 μg/mL), rabbit anti-mouse Id1 antibody (CalBioreagents, San Mateo, CA, USA, 10 μg/mL), or purified nonspecific mouse and rabbit immunoglobulin G (IgG) (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C in blocking buffer. After washing, tissues were incubated with a biotinylated anti-mouse or anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA, 10 μg/mL) for 1 h at 37 °C in blocking buffer. Vectastain ABC kit (Vector Laboratories) was used to detect the antibodies on the tissues, following manufacturer’s protocols. Sections were mounted with Cytoseal 60 (Thermo Fisher Scientific), visualized under an Olympus microscope (Olympus, Tokyo, Japan) and scored by a pathologist.

Tissue slides were fixed in cold acetone for 20 minutes. Following incubation with 3% H₂O₂ for five minutes to block endogenous peroxidase, STs were blocked with 20% fetal bovine serum (FBS) and 5% goat serum in phosphate-buffered saline (PBS) at 37°C for one hour, and then incubated with mouse anti-human Id1 antibody (Abcam, 10 μg/ml), rabbit anti-mouse Id1 antibody (Cal Bioreagents, San Mateo, CA, USA) or purified nonspecific IgG for one hour at 37°C in blocking buffer. The ST samples were washed with PBS, and a 1:200 dilution in blocking buffer of biotinylated goat anti-mouse or anti-rabbit antibody was added and incubated for an additional 30 minutes at 37°C. After washing, antibody binding was detected using a Vectastain ABC Elite kit (Vector Labs) and the chromogen 3,3′-diaminobenzidine (DAB) (Vector Labs). ST samples were counterstained with Harris hematoxylin. Staining was evaluated by a pathologist who was blinded with regard to the sample group. Slides were examined for cellular immunoreactivity, and cell types were distinguished based on their characteristic morphology. The percentage of cells expressing Id1 was analyzed and graphed.

Rheumatoid factor (RF) was depleted from human SFs using anti-human IgM (μ-chain specific) agarose antibody (Sigma-Aldrich, St. Louis, MO, USA). Levels of Id1 were measured using 96-well plates. RA, OA and other disease SFs, and Id1 as a standard were coated in duplicate for one hour. The plates were washed with wash buffer and coated with blocking buffer. Mouse anti-human Id1 antibody (Abcam) in blocking buffer was added for one hour. Subsequently, biotinylated goat anti-mouse antibody (Vector Labs, Burlingame, CA, USA) and streptavidin-HRP (BD Biosciences, San Jose, CA, USA) were added, and the concentration in samples was measured at 450 nm after developing the reaction with tetramethylbenzine substrate (TMB, Sigma-Aldrich). For the CXCL16 ELISA, 96-well plates were coated with rabbit anti-human CXCL16 (PeproTech, Rocky Hill, NJ, USA). SFs and rhuCXCL16 (PeproTech) as a standard were added. Biotinylated rabbit anti-human CXCL16 antibody (PeproTech) was used to detect CXCL16 using a streptavidin-HRP, with TMB. The concentration in each sample was measured at 450 nm.