Immunofluorescence Analysis of Muscle Tissue

The muscles were cut on a cryostat at − 23 °C (7 μm), air-dried, and stored at − 20 °C. Slides were air-dried, rehydrated, and fixed in 4% paraformaldehyde (PFA) for 20 min at the time of staining. For CD63/DAPI/laminin staining, sections were incubated with mouse anti-CD63 IgG1 antibody (1:100 dilution, ab108950, Abcam, Cambridge, UK) and rabbit anti-laminin IgG antibody (1:100 dilution, L9393, Sigma-Aldrich, St. Louis, MO) overnight at 4 °C. Slides were washed in PBS, then incubated with Alexa Fluor 488 goat anti-mouse IgG1 (1:250 dilution, A11001, Invitrogen, Waltham, MA) and Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilution, A11012, Invitrogen) secondary antibodies for 1 h at room temperature. Slides were washed in PBS and mounted with VectaShield fluorescent mounting media with DAPI (H-1200-10, Vector Laboratories, Newark, CA). For CD9/DAPI/dystrophin staining, sections were incubated with rabbit anti-CD9 IgG (1:100 dilution, SA35-08, Invitrogen) and mouse anti-dystrophin IgG2b (1:250 dilution, 08168, Sigma-Aldrich) overnight, followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilution, A11012, Invitrogen) and Alexa Fluor 647 goat anti-mouse IgG2b (1:250 dilution, A32728, Invitrogen) for 1 h at room temperature. For CD81/DAPI/dystrophin staining, sections were incubated with rabbit anti-CD81(1:100 dilution, SN206-01, Novus Biologicals, Centennial, CO) and mouse anti-dystrophin IgG2b (1:250 dilution, 08168, Sigma-Aldrich) overnight, followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilution, A11012, Invitrogen) and Alexa Fluor 647 goat anti-mouse IgG2b (1:250 dilution, A32728, Invitrogen) for 1 h at room temperature. For Pax7/CD9/DAPI/WGA staining, sections were subjected to epitope retrieval using sodium citrate (10 mM, pH 6.5) at 92 °C, followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide in PBS. Sections were incubated overnight in mouse anti-Pax7 IgG1 (1:100 dilution, Developmental Studies Hybridoma Bank, Iowa City, IA) and rabbit anti-CD9 IgG (1:100 dilution, SA35-08, Invitrogen), followed by incubation in goat anti-mouse biotin-conjugated secondary antibody (dilution 1:1,000, 115-065-205; Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 647 goat anti-rabbit IgG (1:250 dilution, A32733, Invitrogen) for 1 h at room temperature. Next, sections were incubated with streptavidin-HRP (1:500 dilution, S-911, Invitrogen) and Texas Red-conjugated Wheat Germ Agglutinin (WGA) (1:50 dilution, W21405, Invitrogen) at room temperature for 1 h, before incubation in Tyramide Signal Amplification (TSA) Alexa Fluor 488 (1:500 dilution, B40953, Invitrogen). Sections were mounted with VectaShield fluorescent mounting media with DAPI (H-1200-10, Vector Laboratories).
Images were captured with a Zeiss upright microscope (AxioImager M1, Oberkochen, Germany). To quantify the percentage of nuclei (DAPI+) expressing CD63, MyoVision software was used for automated analysis of nuclear density in cross-sections [39 (link)], and nuclei-expressing CD63 (identified as DAPI+/CD63+ events) were counted manually in a blinded manner by the same assessor for all sections using the Zen Blue software.