Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers a
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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 Mélina Bailly1,2 · Léonard Féasson3 · Bruno Pereira4 · Amandine Boileau1 · Christophe Hourdé5 · Natacha Germain2,6 · Bogdan Galusca2,6 · Daniel Courteix1 · David Thivel1 · Julien Verney1 Accepted: 16 June 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. Keywords Capillary supply · Immunohistochemistry · Combined staining method · Muscle fiber type · CD31 * Julien Verney [email protected]
David Thivel [email protected]
Mélina Bailly [email protected]
1
Léonard Féasson leonard.feasson@univ‑st‑etienne.fr
Université Clermont Auvergne, CRNH, AME2P, 63000 Clermont‑Ferrand, France
2
Eating Disorders, Addictions and Extreme Bodyweight Research Group (TAPE) EA 7423, Jean Monnet University, Saint‑Étienne, France
3
Inter‑university Laboratory of Human Movement Biology (LIBM) EA 7424, Jean Monnet University, Saint‑Étienne, France
Christophe Hourdé christophe.hourde@univ‑smb.fr
4
Biostatistics Unit, Délégation à la Recherche Clinique et à l’Innovation (DRCI), Clermont‑Ferrand, France
Natacha Germain natacha.germain@chu‑st‑eti
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