A newly modified isolation method of single muscle fibers--especially useful in histological, histochemical and electron microscopic studies on branched fibers

The nitric acid muscle fiber digestion method has generally been used to determine total numbers of muscle fibers and branched fibers. However, it is not suitable for further studies because nitric acid causes protein denaturation known as the xanthoproteic reaction which make it difficult to examine the fiber types and morphology of muscle fibers. Therefore, we attempted to modify this method to preserve the morphology of muscle fibers as much as possible. Our modifications were as follows: (1) samples were immersed in ion exchanged water before nitroc acid treatment; (2) mammalian relaxing solution was used as the post- incubation solution; (3) the temperature of nitric acid was maintained at 4 degrees C; and (4) the nitric acid concentration was reduced to 10% from 15%. The samples obtained by this method were stained with phosphotungstic acid-hematoxylin and their striations were examined by a differential interference contrast method using light microscopy. The cell organella i.e. actin filaments, myosin filaments, T-tubules and mitochondria, were also examined electron microscopy. Actin and myosin filaments in these samples were also stained immunohistochemically to clarify the preservation of antigenicity. As a result, this modified method made it possible to examine actin and myosin filaments of a single muscle fiber light-microscopically and immunohistochemically and also to examine cell organella of a single muscle by electron microscopy. These results indicate that our method is useful for studies on branched muscle fibers such as stereological analysis and innervation of a single branched muscle fiber, in addition to obtaining muscle fiber numbers.