Ab193955

sfd-FLSs were homogenized using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentration was measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Subsequently, protein samples (20 µg in each lane) were separated by 12.5% SDS-PAGE. Protein were blotted on a nitrocellulose membrane and the membranes were incubated with primary antibodies anti-IL-17 (1:1,000; ab193955; Abcam), TNF-α (1:1,000; ab109332; Abcam), ERK (1:1,000; ab32537; Abcam), pERK (1:1,000; ab201015; Abcam), STAT1 (1:1,000; ab2071; Abcam), pSTAT1 (1:1,000; ab30645; Abcam) and β-actin (1:1,000; ab8226; Abcam) for 12 h at 4°C, after blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. PV-6001; OriGene Technologies, Inc.) for 24 h at 4°C. The blots were visualized using an enhanced chemiluminescence detection system (cat. no. 32209; Pierce; Thermo Fisher Scientific, Inc.). Densitometric quantification was performed using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc.).

Synovial membranes were collected from rats 28 days after collagen injection. Tissues were fixed with 4% paraformaldehyde at room temperature for 12 h. Paraffin-embedded tissue samples of synovial membranes were obtained and cut into 4 µm sections, deparaffinized and rehydrated using a descending alcohol series. Sections were prepared and epitope retrieval was performed using Tris-HCl buffer (cat. no. AP-9005-050; Thermo Fisher Scientific, Inc.) for 30 min at 37°C. Tissue sections were stained H&E (Sigma-Aldrich) for 15 min at room temperature. Sections were treated with 3% hydrogen peroxide for 15 min at 37°C and subsequently blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Sections were washed with PBS and incubated with rabbit anti-rat IL-17 (1:1,000; ab193955; Abcam), TNF-α (1:1,000; ab109332; Abcam), ERK (1:1,000; ab32537; Abcam), phosphorylated ERK (pERK; 1:1,000; ab201015; Abcam) and STAT1 (1:1,000; ab2071; Abcam) at 4°C overnight. Sections were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:2,000; cat. no. 1706515; Bio-Rad Laboratories, Inc.) for 1 h at 37°C. Diaminobenzidine was used as substrate for the immunohistochemical reaction. Tissue sections were visualized at ×200 magnification using a confocal microscope (LSM780; Carl Zeiss AG).