Laminin antibody

Left triceps muscles were snap frozen in 2-methylbutane (Sigma-Aldrich) cooled in liquid nitrogen. Sections of 8 μm were made on SuperFrost Ultra Plus slides (Thermo Fisher Scientific, Germany) with a Leica CM3050 S cryostat (Leica Microsystems, the Netherlands). Slides with frozen muscle sections were warmed up for 30 min at room temperature, fixated in ice-cold acetone for 5 min, and air-dried for 30 min. A circle was drawn around each muscle section with a DAKO pen (Dako Denmark, Denmark) and washed with PBS. Subsequently, slides were stained overnight at 4°C with primary antibody (Dystrophin antibody, 1:50 [Santa Cruz, Germany]; sc-7461; Laminin antibody, 1:100 [Abcam, UK]; ab11575) in blocking buffer (PBS/0.05% Tween/5% horse serum). The next day slides were washed three times with PBS and incubated with labeled secondary antibody for 60 min at room temperature in the dark (Alexa 594 red [for laminin], 1:1,000 [Life Technology, the Netherlands]; Alexa 488 green [for dystrophin], 1:1,000 [Life Technology, the Netherlands]). Subsequently, slides were washed three times with PBS and embedded in ProLong Gold Antifade Mounting medium. Slides were dried overnight at 4°C and analyzed using a BZ-X700 fluorescent microscope (Keyence, Japan), 10× magnification.

The TA muscle of mice was fixed with 4% paraformaldehyde. The fixed muscle tissue was dehydrated by gradient sucrose solution, and then chopped and cut into 10 µm thick sections on the quick-freezing table. The sections were then placed on the adhesive slide and left overnight at room temperature for drying. The slides to be dyed were washed with PBS three times for 10 minutes and blocked with blocking solution for 1 hour. To determine the cross-sectional area (CSA) of skeletal muscle fiber and the content of fast twitch muscle fiber, slices were incubated with laminin antibody (Abcam, Cambridge, UK; dilution 1:200) and fast myosin skeletal heavy chain antibody (Abcam; dilution 1:200) at 4 °C for 12 hours, respectively. Then the slides were incubated with secondary antibody Alexa Fluor (Invitrogen Antibodies, Waltham, MA, USA) at room temperature for 60 minutes. Finally, the slides were photographed with a fluorescence microscope (Zeiss, Germany) to obtain the CSA of skeletal muscle fiber and the content of fast twitch muscle fiber.