Axiovision ac release 4

Serial sections (5 µm) were cut transversely through the TA muscle using a refrigerated (−20ºC) cryostat (CTI Cryostat; IEC, Needham Heights, MA, USA). Sections were reacted with laminin (Sigma-Aldrich) for determination of myofiber cross-sectional area (CSA) and with SC-71 and BF-F3 (both developed by S. Schiaffino, University of Padova, obtained from the Developmental Studies Hybridoma Bank) to assess the percentage of myosin IIa and myosin IIb isoforms, respectively (24 (link)). We have previously shown that mouse TA muscles do not express detectable levels of type I fibers (25 ) and so the fibers not reacting with SC-71 or BF-F3 were assumed to be type IIx fibers. Digital images were obtained using an upright microscope with camera (Axio Imager D1, Carl Zeiss), controlled and quantified using AxioVision AC software (AxioVision AC release 4.8.2, Carl Zeiss).

Cells were washed for 2 × 5 min in PBS and fixed in 4% paraformaldehyde for 15 min. Cells were then washed in PBS (3 × 5 min), permeabilized with 0.1% Triton X-100 for 10 min, washed in PBS (3 × 5 min) and blocked in 3% (w/v) BSA in PBS for 1 h at RT. Cells were incubated overnight at 4ºC in anti-sarcomeric myosin (1:50 diluted in 3% BSA/PBS, MF 20, developed by D.A. Fischman, Weill Cornell Medical College, obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA, USA). The following day, cells were washed in PBS (4 × 5 min) and incubated in goat-anti-mouse Alexa Fluor 555 secondary antibody (1:400, Life Technologies) and DAPI (1:1,000) for 2 h at RT. Cells were washed in PBS (4 × 5 min) and then imaged on a Zeiss Axiovert 40 CFL inverted microscope using a 20× objective to give a total magnification of 126×. Five images were taken in each well from pre-defined locations within each quadrant. Myotube diameter was quantified using AxioVision AC software (AxioVision AC release 4.8.2, Carl Zeiss, Wrek, Göttingen, Germany).