Fibre typing using sarcoplasmic reticulum Ca2+-ATPase and myoglobin immunohistochemistry in rat gastrocnemius muscle

Abstract

Skeletal muscle fibre types were identified by using immunohistochemical detection of sarcoplasmic reticulum Ca2+-ATPase and myoglobin content in rat gastrocnemius muscle. The strong Ca2+-ATPase-reactive fibres were identical with the fast-twitch population, while the fibres with weak reactivity represented the slow-twitch type. Strong myoglobin immunoreactivity reflected the fast oxidative glycolytic (FOG) and slow oxidative (SO) types. Slight to moderate myoglobin immunostaining was found in the fast glycolytic (FG) fibres. The staining intensity of the different fibre types differed as follows: for Ca2+-ATPase FG greater than FOG greater than SO, and for myoglobin FOG greater than SO greater than FG. The immunoreactivity of Ca2+-ATPase and myoglobin were well preserved after fixation of the muscles in Bouin's solution, or in formol/acetic acid fixative, and paraffin embedding. Detection of the primary antibodies was carried out by using the avidin-biotin-peroxidase complex, and the immunogold-silver-staining methods. The latter was found to be more sensitive and suitable for postembedding ultrastructural demonstration of the Ca2+-pump enzyme on Durcupan-embedded muscles. The method, using 5 nm immunogold conjugate with silver enhancement, offered the advantages of high sensitivity and excellent visualization of the reaction product. The postembedding detection of sarcoplasmic reticulum Ca2+-ATPase also proved to be useful in the retrospective identification of the main fibre classes in human muscle biopsies.