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.
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Acknowledgments
The A4.951, A4.840, and N2.261 monoclonal antibodies developed by H. M. Blau were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. The 212F mAb was produced in-house from hybridomas generously donated by Dr. Peter Merrifield, University of Ontario, Canada. We are grateful to the staff of the Metabolic Research Unit at the Jean Mayer USDA HNRCA at Tufts University for assistance in carrying out on this study. LC is supported by NIH KL2 RR025751. The study was funded by the Jean Mayer USDA HNRCA at Tufts University and P30 AG031679. None of the authors had any conflicts of interest. This material is based upon work supported by the U.S. Department of Agriculture, Agricultural Research Service, under agreement No. 58-1950-7-707. Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Dept of Agriculture.
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Ceglia, L., da Silva Morais, M., Park, L.K. et al. Multi-step immunofluorescent analysis of vitamin D receptor loci and myosin heavy chain isoforms in human skeletal muscle. J Mol Hist 41, 137–142 (2010). https://doi.org/10.1007/s10735-010-9270-x
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DOI: https://doi.org/10.1007/s10735-010-9270-x