Immunohistochemistry

Protein expression was evaluated on 7 μm tissue sections of ankle joints. Sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by 3% (v/v) hydrogen peroxide in methanol for 20 minutes at room temperature (RT). Sections were washed in between incubation steps with phosphate-buffered saline (PBS) 3 times for 5 minutes. For all sections, a biotin-streptavidin horseradish peroxidase detection system was used according to manufacturer’s protocol (Vector Laboratories; PK6101). Bound complexes were visualized with diaminobenzidine (DAB) (Merck) for 10 minutes at RT. Sections were counterstained with hematoxylin for 30 seconds. Sections were dehydrated and enclosed with Permount.

For F4/80 detection, antigen-retrieval was performed in citrate buffer (pH 6.0) heated to 37ºC for 2 hours and subsequent trypsin treatment (Sigma; T-9201; 0.075% in PBS) at 37°C for 7 minutes. Sections were blocked by 10% (v/v) rabbit serum in PBS for 20 minutes at RT. Tissue sections were incubated with rat anti-mouse F4/80 (eBioscience; 14-4801-81; monoclonal (BM8); 1 μg/mL) or rat IgG2a (BD Pharmingen; 553927; 1 μg/mL) overnight at 4°C. Subsequently, sections were incubated with biotinylated rabbit anti-rat IgG (Vector Laboratories; BA-4001; 1:200) for 30 minutes at RT. Antibodies were diluted in 2% (v/v) rabbit serum in PBS.

For CD3 detection, antigen retrieval was performed in citrate buffer (pH 6.0) heated to 60ºC for 10 minutes. Sections were blocked by 5% (w/v) skimmed milk (ELK) 3% (v/v) fetal calf serum (FCS) 2% (w/v) bovine serum albumin (BSA) in PBS for 1 hour at RT. Tissue sections were incubated with rabbit anti-mouse CD3 (DAKO; A0452; polyclonal; 3 μg/mL) or rabbit IgG control (DAKO; X0936; 3 μg/mL) overnight at 4°C. Subsequently, sections were incubated with biotinylated goat anti-rabbit IgG (PK6101; 1:400) for 30 minutes at RT. Antibodies were diluted in 5% ELK 3% FCS 2% BSA in PBS.

For CD19 detection, antigen retrieval was performed in citrate buffer heated (pH 6.0) to 60ºC for 10 minutes. Sections were blocked by 5% ELK 3% FCS 2% BSA in PBS for 1 hour at RT. Tissue sections were incubated with rabbit anti-mouse CD19 (Abcam; ab203615; polyclonal; 0.4 μg/mL) or rabbit IgG control (DAKO; X0936; 0.4 μg/mL) overnight at 4°C. Subsequently, sections were incubated with biotinylated goat anti-rabbit IgG (PK6101; 1:400) for 30 minutes at RT. Antibodies were diluted in 5% ELK 3% FCS 2% BSA in PBS.