Experimental Cell Research 67 (1971) l-10 MYOGENESIS IN VITRO AS DEMONSTRATED BY IMMUNOFLUORESCENT STAINING WITH ANTIMUSCLE SERUM R. L. DAWKINS and MARGARET LAMONT MRC Rheumatism Research Unit, Taplow, Maidenhead, Berks, dnd Department of Experimental Pathology, Charing Cross Hospital Medical School, Hammersmith, London W 6, UK SUMMARY Immunofluorescent staining of trypsinized chick muscle cultures with a guinea pig anti-rabbit- muscle serum has allowed specific identification of muscle cells at an earlier stage than has previously been reported. When filtrates of trypsinized muscle were stained rounded multinucleated cells were seen. When sequential cultures were examined it was found that these multinucleated cells devel- oped into typical myotubes within 18 h. Mononucleated myoblasts were stained after 9 h in culture; these apparently fused and subsequently followed the well recognised pattern of myo- genesis. Cross-striations were stained after only 18 h. These results indicate that both multinucleated and mononucleated cells can contribute to myogenesis after trypsinization. Although immunofluorescence has been used in the study of myogenesisin vitro [I 11, the technique has not been successfully applied to the elucidation of the events which occur immediately after initiating culture. Holtzer and co-workers [.5, 9, IO] stained cultures of trypsinized chick muscle with fluorescein-labelled antisera from rabbits immunised with a preparation of chick myosin. In the first two days most staining was extracellular or within moribund cells. A small number of apparently normal myo- blasts were stained at this stagebut it was not until after 2 or 3 days that normal myo- blasts and myotubes were stained selectively and in large numbers. Engel & Horvath [3], in contrast, used myosin prepared from r Present address: Department of Pathology, Uni- versity of Western Australia, Victoria Square, Perth, Western Australia, 6000. l-711811 human or cat muscle to raise their antimyo- sin. Using explant cultures of chick muscle these workers observed specific staining of myoblasts and myotubes after 24-48 h but interpretation was complicated by staining of direct extensions from the explant. From theseresults it would seem that there is a need for more sensitive and more specific staining of muscle in early cultures. We have therefore used a modified approach in- corporating the following techniques: (i) indirect immunofluorescence; (ii) antiserum raised against crude muscle rather than myosin in view of the possibility that such an antiserum could react with muscle-spe- cific antigens which differentiate before myosin; (iii) fixation methods likely to pre- serve as full an antigenic complement as possible; (iv) antiserum raised against muscle from a different species to that in culture so ExptI Cell Res 67