Muscle Cryosectioning and Laminin Imaging

Quadriceps muscles were embedded in Optimal Cutting Temperature compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen-cooled 2-methylbutane (Sigma, St. Louis, MO, USA), and stored at -80° C. Transverse 7 μm-thick frozen sections were cut with a Leica cryostat and mounted onto SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). Muscle sections were dried for 2 hours at -20° C, and then stored at -80° C until analysis. Frozen quadriceps sections were removed from -80° C and fixed in 4% paraformaldehyde (PFA) for 15 minutes. After being permeabilized and blocked, the sections were incubated with rabbit anti-laminin antibody (L9393, Sigma-Aldrich) overnight at 4° C, followed by goat-anti-rabbit cross-adsorbed secondary antibody Alexa Fluor 488 (A11008, Invitrogen by Thermo Fisher Scientific) for 1 hour in a humidified chamber at room temperature. Sections were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen by Thermo Fisher Scientific). Images were captured at 10x magnification with a Zeiss Axio Imager microscope. Myofiber cross-sectional area, demarcated by Laminin staining, was quantified using MuscleJ [68 (link)].