Immunohistochemical Investigations of Parvalbumin Localization in the Skeletal Muscle Fibers of Rats

Abstract

Parvalbumin (PA) is a water soluble, low-molecular weight, calcium-binding protein which has been thought to be involved in the relaxation of skeletal muscle fibers. Although it is well known that PA concentrations are higher in fast twitch fiber than slow twitch fiber, the localization of PA within the cytoplasm of single muscle fibers is still unknown. The present study, therefore, was undertaken to clarify the PA localization by immunohistochemical methods using the confocal laser scanning microscope (CLSM) and transmission electron microscope (TEM).
Wistar strain male rats were fixed by vascular perfusion with 4% paraformaldehyde solution, and the extensor digitorm longus muscle was dissected out. For fluorescent antibody examination, these specimens were quickly frozen in melting isopentan and sections were cut using cryestat at-25°C. These sections were incubated in anti-PA and anti-Troponin (TR) respectively, and then exposed to Texas-Red- and FITC-labelled secondary antibodies. For TEM study, the pre-embedding method was used. Fluorescent immunohistochemical study has clearly shown that both PA and TR are located intimately in the I-band of the skeletal muscle fibers. The finding by the immunofluorescent study correlated well with those which have been seen at the ultrastructural level. The fact that PA is located in close proximity to TR is considered to be very reasonable when we consider it in terms of the muscular contraction-relaxation cycle.