Anti il 17a

Human MSG tissues from 10 pSS patients and 7 Sicca control patients were obtained after informed consent. The gland tissues were fixed with 4% paraformaldehyde, followed with embedding in paraffin blocks. The slides were heated at 65°C for 30 min, followed by paraffin removal with xylene and subsequent rehydration with ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) at a sub-boiling temperature for 20 min. Samples were blocked with 10% goat serum for 1 h at RT and incubated with primary antibody (1:100) overnight at 4°C. The slides were visualized using streptavidin peroxidase IHC assay kit (ZSGB-bio, China) and counter stained with hematoxylin. The primary antibody of immunohistochemistry was performed using anti-IL-17A (Clone G-4, Santa Cruz), anti-CD4 (Clone EPR6855, Abcam), anti-p-STAT3 (Clone EP2147Y, Abcam)m and anti-p-STAT5 (Clone E208, Abcam). Images were obtained using an OLYMPUS-BX51 microscope at 10 × 10 or 40 × 10 magnification.

Bone marrow samples obtained from six OA patients and six RA patients were examined histopathologically. BM samples were fixed in Oxford fixative (formaldehyde 40%, glacial acetic acid 2%, sodium chloride 8.7%, distilled water), routinely processed and embedded in paraffin wax. Sections 3 μm thick were cut and stained with haematoxylin and eosin. The following antibodies were then used for further staining: anti-CD8 (polyclonal Ab, dilution 1:50; Dako, Glostrup, Denmark), anti-CD4 (clone 4B12, dilution 1:10; Novocastra, now part of Leica Microsystems, Wetzlar, Germany) and anti-IL-17A (dilution 1:50; Santa Cruz). Staining was performed according to the manufacturer’s instructions. The EnVision Detection System (Dako Denmark A/S, Glostrup, Denmark) was used for detection. Positive controls were performed on human tonsils. Negative (isotype) controls were performed using ready-to-use FLEX Negative Control Mouse (cocktail of murine IgG1, IgG2a, IgG2b, IgG3 and IgM, code number IR750; Dako Denmark A/S). Samples were reviewed for expression of these proteins by a qualified histopathologist who was blinded to outcome. Appropriate cellular localization for immunostaining was membrane for CD8 and CD4 and cytoplasmatic for IL-17A. All photographs were taken using Olympus microscope cameras: DP72 Olympus BX63 and DP12 Olympus BX (Olympus, Tokyo, Japan).