Expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and Fn14 in patients with polymyositis (PM)/dermatomyositis (DM) and in healthy controls. (A) Muscle biopsies from patients with PM/DM showed significantly higher levels of the TWEAK protein compared to those of healthy controls. Anti-human TWEAK polyclonal antibodies were used as the primary antibody for detection at a working concentration of 15 μg/ml. Rhodamine-conjugated anti-goat IgG was used as a second antibody. (B) In PM/DM biopsies, expression of the Fn14 protein was remarkably higher than that of healthy controls. Anti-human Fn14 monoclonal antibodies were used as the primary antibody for detection at a working concentration of 15 μg/ml. Rhodamine-conjugated anti-goat IgG was used as a second antibody. Each muscle section was analyzed in duplicate, and results from one representative experiment are shown. Negative controls were conducted by staining muscle biopsies with isotype control antibody, and no obvious immunofluorescence signal was observed in negative controls. (C) Serial muscle biopsy sections of PM/DM patients were applied for TWEAK immunofluorescence staining and H&E staining, and a representative picture of the same type of field is shown. (D) Muscle biopsy sections of PM/DM patient were applied for TWEAK and Fn14 immunofluorescence staining and a representative staining is shown. To ensure comparability, images were obtained using identical exposure settings for each channel. The TWEAK immunoreactivity is predominantly observed around the infiltrating monocytes, and a mild expression of TWEAK on the sarcolemma of muscle fibers is also visible. While Fn14 are mainly expressed on the sarcolemma of muscle fibers. Therefore, TWEAK and Fn14 are frequently found to be coexpressed in the same myofibers on overlay images. Magnification is 200 ×.