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Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle

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Abstract

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin’s concordance correlation coefficients and Bland and Altman’s graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

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Acknowledgements

The authors thank all the participants of this research.

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Authors

Contributions

JV: Conceptualization, JV, DT, LF, CH, DC, NG, BG: Data curation, project administration, resources, visualization, supervision, JV, MB: Writing—original draft, review and editing, MB, AB, JV: Methodology, Investigation, Validation, BP: Statistical analysis.

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Correspondence to Julien Verney.

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The authors declare that they have no competing interest.

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The present study used muscle samples from a previous study (Galusca et al. 2018) that was conducted in accordance with the Helsinki Declaration and that was approved by the local research and ethics committee of Saint-Étienne, France.

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All subjects gave written informed consent prior to inclusion in the study.

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Bailly, M., Féasson, L., Pereira, B. et al. Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle. Histochem Cell Biol 154, 327–337 (2020). https://doi.org/10.1007/s00418-020-01895-5

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  • DOI: https://doi.org/10.1007/s00418-020-01895-5

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