Ab11576

Fresh-frozen TA were sliced into 10 μm thick sections by a cryostat. The sections were reacted with 100-fold diluted polyclonal anti-dystrophin (cat #ab15277, Abcam, Cambridge, UK) or 1000-fold diluted polyclonal anti-Laminin 2 alpha (cat #ab11576, Abcam, Cambridge, UK) for 2 h at room temperature or overnight at 4 °C and then reacted with goat anti-Rabbit IgG (H;L) antibody conjugated with Alexa568 (cat #A11036, ThermoFisher, 200-fold dilution) for dystrophin detection or biotinylated IgG antibody (cat #BA-4000, Vector laboratories, 200-fold dilution) for laminin 2 alpha detection as the secondary antibody and ABC complex (Vector laboratories, CA) for 1–2 h at room temperature. The numbers of dystrophin fibers and central nuclei were counted by using HALO (Indica labs, Albuquerque, NM).

Rabbit polyclonal antibodies were as follows: MyoD c-20 (sc-304) from Santa Cruz; laminin (ab11575) from Abcam; mouse monoclonal antibodies were as follows: Pax7, myosin heavy chain (human fast fibers; A4.74) and myosin heavy chain embryonic (A1.652) from Developmental Studies Hybridoma Bank (DSHB). Rat monoclonal antibodies against laminin-α2 (ab11576) and rabbit polyclonal antibodies against laminin (ab11575) were from Abcam.

Tissue sections were incubated with anti-Fhl2 antibody (1:400, ab202584, Abcam, Cambridge, UK) and anti-Lama2 antibody (1:400, ab11576, Abcam, Cambridge, UK) at 4 °C overnight. After incubation with secondary antibodies for 2 h, the sections were sealed with antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, 041221210930, Beyotime, Shanghai, China). Images were acquired by a Zeiss AX10 microscope (Carl Zeiss, Weimar, Germany).

TA muscle tissue isolation and freezing were performed as described previously^{11 (link)}. The muscle was frozen in melting isopentane and stored at −80 °C until further analysis. Sections of 8-μm thickness were stained with hematoxylin and eosin (H&E). Composite brightfield images of the complete TA muscle were acquired using Axio Scan.Z1 microscope and ZEN imaging software (Carl Zeiss Microscopy). Additional labeling of muscle sections was completed by combinations of antibodies and probes including DAPI (1 µg/ml; Molecular Probes D21490), monoclonal anti-mouse CD68 (1 µg/ml; Biorad MCA341R), monoclonal anti-rat laminin (2.5 µg/ml; Abcam ab11576), and polyclonal anti-goat GFP (1 µg/ml; Abcam ab6673). Appropriate isotype-specific-conjugated secondary antibodies (1:200; Molecular Probes A21125, A11055, or A21202) were then applied. Fluorescent imaging of the TA muscle was conducted with an Olympus FluoView FV1000 laser scanning confocal microscope (Olympus America Inc., Melville, NY) mounted on an inverted Olympus IX81 microscope and equipped with Multi-line Argon (458, 488, and 515 nm), HeNeG (543 nm), diode (405 nm), and diode (635 nm) lasers using an Olympus UPLSAPO 20×/0.85 N.A. oil immersion lens. All images were acquired as 12-bit multi-TIFF files.