Quantifying Muscle Angiogenesis in Ischemic Mice

Mice were sacrificed 14 d after FAL to collect ischaemic left gastrocnemius muscle. 7-μm thick cryosections were first blocked with 5% normal goat serum and then incubated with primary antibodies CD31 (a marker for endothelial cell, Santa Cruz, CA) and α-smooth muscle actin (α-SMA) (Abcam, UK, Cambridge) at 4 °C overnight, followed by the incubation with secondary antibodies Alexa-488 conjugated anti-rabbit IgG and Alexa-594 conjugated anti-mouse (1:500; Jackson ImmunoResearch Laboratories, PA, USA) for 1 h at room temperature. Subsequently, sections were mounted using Vectashield with DAPI (4'6-diamino-2-fenilindol dihidrocloreto) for counter-staining of nuclei before observation. Pictures from each section were taken under 400× magnification, using Olympus BX51 high-magnification microscope. Capillaries were identified by positive staining for CD31 and those displaying a second cellular layer stained with SMA, surrounding the inner one, were counted as arterioles. According to the procedures previously published [25 (link),26 (link)], ten different fields from each tissue section were selected randomly. Capillaries labelled with CD31 were counted. Capillary density was expressed as the number of capillaries per square millimetre. The proportion of fibres with central nucleus (regenerated fibres) in the injured area was calculated [27 (link)].