Skeletal Muscle Fiber Analysis

Laminin and haematoxylin and eosin (H & E) staining were performed as previously described [47 (link),51 (link)]. To measure the cross sectional area (CSA) of myofibres, muscle sections were stained with an anti-laminin A antibody (L1293; Sigma-Aldrich). Laminin, a cell-adhesion molecule strongly expressed in the basement membrane of skeletal muscle, was detected using an appropriate secondary antibody. Morphometric analyses were performed on sections collected from similar regions of each muscle using a Leica DMI4000 B automated inverted microscope equipped with a DCF310 digital camera. Image acquisition was controlled by the Leica LAS AF software. The ImageJ software was used to determine the CSA of 1,000 to 3,000 individual fibres from at least two different fields for each muscle section. Four to nine sections from each muscle were analysed. For histological analyses, serial muscle sections were obtained and stained in H & E following standard procedures. The number of fibres was counted and analysed using the ImageJ software.
Single myofiber isolation of hind limb muscle and nuclei immunofluorescence on single fibers was performed as previously described [8 (link)]. Nuclei of 30 individual fibres from each muscle were analysed.