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The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Background: The quantitative analysis of muscle histomorphometry has been growing in importance in both research and clinical settings. Accurate and stringent assessment of myofibers' changes in size and number, and alterations in the proportion of oxidative (type I) and glycolytic (type II) fibers is essential for the appropriate study of aging and pathological muscle, as well as for diagnosis and follow-up of muscle diseases. Manual and semi-automated methods to assess muscle morphometry in sections are time-consuming, limited to a small field of analysis, and susceptible to bias, while most automated methods have been only tested in rodent muscle., Methods: We developed a new macro script for Fiji-ImageJ to automatically assess human fiber morphometry in digital images of the entire muscle. We tested the functionality of our method in deltoid muscle biopsies from a heterogeneous population of subjects with histologically normal muscle (male, female, old, young, lean, obese) and patients with dermatomyositis, necrotizing autoimmune myopathy, and anti-synthetase syndrome myopathy., Results: Our macro is fully automated, requires no user intervention, and demonstrated improved fiber segmentation by running a series of image pre-processing steps before the analysis. Likewise, our tool showed high accuracy, as compared with manual methods, for identifying the total number of fibers (r = 0.97, p < 0.001), fiber I and fiber II proportion (r = 0.92, p < 0.001), and minor diameter (r = 0.86, p < 0.001) while conducting analysis in ~ 5 min/sample. The performance of the macro analysis was maintained in pectoral and deltoid samples from subjects of different age, gender, body weight, and muscle status. The output of the analyses includes excel files with the quantification of fibers' morphometry and color-coded maps based on the fiber's size, which proved to be an advantageous feature for the fast and easy visual identification of location-specific atrophy and a potential tool for medical diagnosis., Conclusion: Our macro is reliable and suitable for the study of human skeletal muscle for research and for diagnosis in clinical settings providing reproducible and consistent analysis when the time is of the utmost importance.