Retrieval of cryostat sections for comparison of histochemistry and quantitative electron microscopy in a muscle fiber

Abstract

A method is presented that can be used to perform histochemical and morphometric analyses on the same muscle fiber. Freshly dissected fibers from medial gastrocnemius muscle of adult guinea pig were kept at a resting length and rapidly frozen. Serial frozen cross- sections were cut and reacted for myofibrillar adenosine triphosphatase and succinic dehydrogenase. The adjacent section, while still frozen, was immersed into 20°C glutaraldehyde fixative to which EGTA was added to minimize artifactious contractions. The fixed section was processed for electron microscopy and the section rotated before thin sectioning to give longitudinal sections enabling study of sarcomeres. Ultrastructure was well-preserved despite slight disorganization of the contractile filaments and some vesiculation of the sarcoplasmic reticulum. The Z line width was measured and the mitochondrial volume fraction estimated by point counting morphometry from 89 fibers. The fibers with dark myofibrillar adenosine triphosphatase staining have Z widths of 547 ± 165 Å (n = 69) and those with light stain have 1023 ± 113 Å (n = 20). The density of the succinic dehydrogenase reaction product in the fibers was divided into dark and light and the mitochondrial volume fractions were found to be 4.3 ± 2.1% (n = 52) and 1.0 ± 1.1% (n = 37) respectively.