Quantifying dystrophin-positive revertant fibers

Q muscles were embedded in optimum cutting temperature medium (OCT; VWR International, Dublin, Ireland) and frozen in liquid nitrogen chilled isopentane (VWR International). A total of 10 µm transversal cryosections were fixed with 4% paraformaldehyde and blocked with 10% goat serum. Antibodies directed against dystrophin and laminin were used (Table S1). Secondary antibodies were AF488 and AF647-conjugated goat anti-rabbit or anti-rat (Sigma-Aldrich, St-Louis, MO, USA), respectively. Finally, nuclei were stained with DAPI (Thermo Fisher Scientific). Whole slides were scanned with a fluorescence microscope (Axio Scan.Z1, Zeiss, Oberkochen, Germany). Dystrophin-positive fibres were counted and expressed as the percentage of the total number of fibres per muscle (ZEN 3.4 blue edition, Zeiss). A dystrophin-positive revertant fibre (RF) was scored when more than half of its membrane circumference expressed a green positive signal [27 (link)]. The number of clusters (with at least two adjacent RFs) and the maximal number of RFs in a cluster were counted on whole sections as well [27 (link)]. Centrally nucleated fibres (CNF) were also calculated using a personalized version of MuscleJ [28 (link)] based on the automatic detection of laminin and DAPI immunofluorescence.

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Dubuisson N., Versele R., Davis-López de Carrizosa M.A., Selvais C.M., Noel L., Planchon C., Van den Bergh P.Y., Brichard S.M, & Abou-Samra M. (2023). The Adiponectin Receptor Agonist, ALY688: A Promising Therapeutic for Fibrosis in the Dystrophic Muscle. Cells, 12(16), 2101.

Publication 2023

DAPI ZEN 3.4 blue edition

Af647 Antibodies Cryosections Dapi Dystrophin Fibres Fluorescence microscope Frozen Goat Immunofluorescence Isopentane Laminin Membrane Muscle Nitrogen Nuclei Paraformaldehyde Rabbit Scan Serum

Corresponding Organization : UCLouvain

Other organizations : Cliniques Universitaires Saint-Luc, Universidad de Sevilla

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Variable analysis

independent variables

Cutting temperature medium (OCT)

dependent variables

Percentage of dystrophin-positive fibres per muscle
Number of dystrophin-positive revertant fibres (RFs)
Number of clusters with at least two adjacent RFs
Maximal number of RFs in a cluster
Percentage of centrally nucleated fibres (CNF)

control variables

10 μm transversal cryosections
4% paraformaldehyde fixation
10% goat serum blocking
Antibodies directed against dystrophin and laminin
AF488 and AF647-conjugated secondary antibodies
DAPI nuclear staining
Fluorescence microscope scanning

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