Multi-color Immunofluorescence Procedure for Rapid Identification of MHC Expression in Human Skeletal Muscle

Abstract

We have previously outlined an immunofluorescence staining protocol for evaluating the fiber type composition of rat and mouse skeletal muscles. The main advantage of this protocol compared to typical myosin ATPase and immunoperoxidase procedures is that multiple myosin heavy chain (MHC) isoforms can be identified on a single cross-section, eliminating the need for multiple incubations. Additionally, detection of MHC expression directly allows for more accurate fiber type identification as differential pH-dependant inactivation of myosin ATPase has been noted across species. However, antibody cross-reactivity with human muscle must also be considered. PURPOSE: To examine the effectiveness of a rapid immunofluorescence fiber-typing procedure on human skeletal muscle. METHODS: Cross-sections of vastus lateralis muscle from eight subjects were incubated with antibodies against MHCI (BA-F8), IIa (SC-71 or 2F7), and IIx (6H1). An additional antibody, specific for all MHC isoforms except IIx (BF-35) was also used. Fiber-type specific oxidative and glycolytic capacity was measured by metabolic enzyme activity staining for succinate dehydrogenase (SDH) and glycerophosphate dehydrogenase (GPD), respectively. RESULTS: Fibers demonstrating strong reactivity with BA-F8 were classified as type I (48.9%). A considerable number of fibers stained intensely for SC-71 and 2F7 and were classified as type IIA (42.8%). A very small subset of fibers stained intermediate for both BA-F8 and SC-71 and were classified as type I/IIA (0.1%). A population of fibers displayed reactivity against 6H1; however, this population also showed intermediate staining for both MHCIIa antibodies. These fibers were identified either as pure type IIX based on their non-reactivity with BF-35 (5.6%), or as type IIAX hybrid fibers based on intermediate staining for both 6H1 and BF-35 (2.6%). Type I fibers were observed as being most oxidative and type IIX fibers as being most glycolytic, with hybrid fibers displaying enzyme activity between their respective pure counterparts. CONCLUSIONS: These results suggest that a multi-color approach using three commercially available antibodies simultaneously is effective for identifying both pure and hybrid fibers in human muscle.