Comprehensive insights into muscle physiology necessitate in-depth analyses of fiber types, yet the laborious nature of dissecting and typing individual fibers has posed significant challenges. This often limits investigations to a scant number of fibers from a handful of participants, casting doubts on the representativeness of both the fiber and participant populations. To surmount these obstacles and enable large-scale, fiber-specific studies, an innovative and rapid technique for high-throughput fiber typing of individually dissected fibers has been developed and named Thrifty (High-Throughput Immunofluorescence Fiber Typing). In the Thrifty approach, 400 fiber segments are affixed to microscope slides with a pre-printed grid system, subjected to antibody probing against myosin heavy chain (MyHC)-I and MyHC-II, and classified under a fluorescence microscope. The effectiveness and expediency of Thrifty are benchmarked against a previously established protocol (dot blot) on a fiber-by-fiber basis, while the purity of fiber pools is verified using the gold standard SDS-PAGE and silver staining. Additionally, a modified Thrifty protocol utilizing fluorescence western blot equipment has been successfully validated. Thrifty exhibits exceptional concurrence with the dot blot protocol, yielding a K value of 0.955 (95% CI: 0.928, 0.982), P<0.001. Both the original and modified Thrifty methods consistently yield type I and type II fiber pools of absolute purity. Impressively, the Thrifty procedure accomplishes the typing of 400 fibers in just under 11 hours, nearly three times faster than the dot blot method. Notably, Thrifty emerges as an inventive and dependable approach, boasting remarkable versatility for swift fiber typing of individual fibers. Consequently, Thrifty stands poised to streamline the generation of extensive fiber pools, thereby paving the way for more comprehensive explorations into the intricate mechanisms governing skeletal muscle physiology.
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