Multi-step immunofluorescent analysis of vitamin D receptor loci and myosin heavy chain isoforms in human skeletal muscle

Abstract

Vitamin D receptors have been shown to be present in human skeletal muscle using different techniques. We developed a multi-staining immunofluorescent method to detect vitamin D receptor expression and co-localize it with myosin heavy chain isoform expression in skeletal muscle biopsies in older female subjects. Serial sections were cut from frozen samples obtained by needle biopsy of the vastus lateralis. Samples were probed with a primary vitamin D receptor monoclonal antibody and then re-probed with a type IIa myosin heavy chain isoform-specific antibody. Independent unfixed sections followed a similar protocol and were probed with type IIx and type I myosin heavy chain isoform-specific antibodies. Immunohistochemistry and fluorescent microscopy co-localized vitamin D receptor loci and myosin heavy chain isoforms in whole skeletal muscle sections. We quantified intranuclear vitamin D receptor staining patterns and number of individual muscle fiber subtypes within a muscle section. Immunohistochemical staining of the vitamin D receptor was confirmed by Western blot using the same monoclonal antibody. This multi-staining immunofluorescent technique allows for measurement of intranuclear vitamin D receptor expression in the context of the specific muscle fiber type profile in a single section. This method can thus be a useful approach to study potential relationships between muscle fiber subtypes and vitamin D receptor expression.

Fig. 1

Composite image (100×) of multi-immunofluorescent staining of MHC isoforms in a human skeletal muscle tissue section. Colorization patterns are: dark green = type I, green/blue = hybrid I/IIa, blue = type IIa, red = type IIx, purple = hybrid IIax, and light green = laminin

Fig. 2

VDR-positive myonuclei in skeletal muscle section at 400× magnification showing: a DAPI staining myonuclei; b co-localization of VDR-positive myonuclei with DAPI; c bright field microscopy

Fig. 3

VDR expression in whole cell lysate of human skeletal muscle is shown using three commercially available primary antibodies. From left to right are VDR (D-6), VDR (333C6a) and VDR (NR1I1). VDR expression was verified by use of human VDR transfected 293T cell lysate from Santa Cruz Biotechnology, Inc