Muscle Fiber Analysis via Immunofluorescence

Experimental methods and CSA analysis were based on previous studies^{42 (link)–44 (link)}. The GAS and TA muscles were cut into 10-μm-thick frozen sections using a cryostat (CM-502, Sakura Finetech). Samples were blocked with 5% goat serum (PCN5000, Thermo Fisher Scientific Japan, Tokyo, Japan) for 1 h, incubated with a primary anti-laminin antibody (L8271, 1:1000, Sigma Aldrich Japan, Tokyo, Japan) for 2 h. After incubation, the cells were washed with 0.1 M phosphate buffer (5 min × 3 times) and incubated with Alexa fluor 488 conjugated secondary antibody (1:2000, A-11008, Thermo Fisher Scientific, MA, USA) for 2 h at room temperature. Images were captured using a confocal laser microscope (FV-3000; Olympus, Tokyo, Japan) and quantified using MyoVision (University of Kentucky). Twelve images were acquired from four sections per muscle for analysis using approximately 4500 to 8000 fibers per group.