Objective To explore the optimal frozen section method for fresh and fixed skeletal muscle tissue, and to lay an experimental foundation for the rapid diagnosis of skeletal muscle diseases and the study of the pathogenesis of skeletal muscle diseases.Methods The tibialis anterior muscle of C57BL/6J mice was extracted, and the fresh tissue was treated by liquid nitrogen direct quick freezing, embedding agent combined with liquid nitrogen quick freezing, and foreign body alkane combined with liquid nitrogen quick freezing. The fixed tissues were pre-treated by direct embedding, embedding agent combined with liquid nitrogen quick freezing. After making frozen sections, HE staining was performed, and TISSUEFAX200 was used for scanning. The cross-sectional area of ice crystals and muscle fibers was calculated to evaluate the effect of different pre-treatment methods. Results The morphology of muscle fiber bundles disappeared and a large number of ice crystal vacuoles were observed in fresh tissues after liquid nitrogen direct freezing and foreign body alkane combined with liquid nitrogen freezing. The embedding agent combined with liquid nitrogen quick freezing ( the surface of the tissue is not in direct contact with liquid nitrogen ) shows that the muscle fiber bundle is complete and dense, and there is no ice crystal. It is a pre-treatment method suitable for fresh tissue. Fixed tissue directly embedded is not easy to slice ; after embedding agent combined with liquid nitrogen quick freezing treatment, the muscle fiber bundle was complete and there was no ice crystal.Conclusions The muscle fiber bundles of fresh and fixed tissues treated with embedding agent combined with liquid nitrogen quick freezing are complete and free of ice crystals, which is convenient for further experiments such as immunohistochemistry and immunofluorescence, and is conducive to the accurate and rapid diagnosis of skeletal muscle diseases and the exploration of the pathogenesis of skeletal muscle diseases.