Immunocytochemical and immunohistochemical staining of aromatase or aromatase combined with calreticulin in human articular cartilage tissue, in primary human articular chondrocytes and in immortalized chondrocytes of cell line C-28/I2. (A) Cultured primary articular chondrocytes react with aromatase antibody as revealed by red staining. (B) Cultured chondrocytes of cell line C-28/I2 react with the aromatase antibody. Control cells do not react. (C) Articular cartilage tissue shows immunoreactivity for aromatase, and the control remains unstained. (D) Localization of aromatase by immunofluorescence. The slides present staining of nuclei by 4′,6-diamidino-2-phenylindole (DAPI) and of cytoplasm with fluorescein isothiocyanate (FITC)–conjugated antibodies to aromatase (antibody) or combined staining of nuclei and aromatase (merged image). Cytoplasm of cultured C-28/I2 cells show positive reaction for aromatase. (E) Localization of aromatase combined with calreticulin by immunofluorescence. The slides present staining of nuclei by DAPI; staining of cytoplasm with FITC-conjugated secondary antibodies against aromatase or calreticulin; and combined staining of nuclei, aromatase and calreticulin (merged image). As shown by single-cell laser-scanning microscopy, staining for aromatase and calreticulin is observed at approximately identical cytoplasmic sites. ER: Endoplasmic reticulum. (F) Primary human articular chondrocytes from knee joint tissue with and without osteoarthritis (OA) show aromatase immunostaining equally in all chondrocytes. Scale bars: 50 μm (A and B), 10 μm (C and E) and 20μm (F).