Perfused and Total Muscle Capillary Density.

Mice received a retro-orbital injection of 50 µL of 1 mg/mL Griffonia simplicifolia lectin (GSL) isolectin B4, Dylight 649 (Vector Laboratories; Cat. No. DL-1208) to fluorescently label α-galactose residues on the surface of endothelial cells of perfused capillaries. Following the injection, animals were returned to their cage and allowed 1–2 hours of free movement prior to euthanasia and muscle harvest. The tibialis anterior muscle was frozen in OCT compound and cryo-sectioned as described above. Transverse muscle sections were fixed with 4% paraformaldehyde (ThermoFisher Scientific, Cat. No. J19943-K2) for ten minutes, and permeabilized with 0.25% triton X-100 (Millipore-Sigma, Cat. No. 93443). Following three washes with PBS, slides were blocked in PBS + 5% goat serum + 1% BSA for 4–6 hours. Total capillaries were labeled with a primary antibody raised against PECAM1 (anti-CD31, Abcam, Cat. No. ab28364, 1:100 dilution in blocking solution) overnight at 4°C. The following day slides were washed with PBS and incubated for one hour at room temperature with Alexa-Fluor555 anti-rabbit secondary antibody (ThermoFisher Scientific, Cat. No. A11034, 1:250 dilution) and wheat germ agglutin (WGA) conjugated with Alexa-Flour488 1mg/ml (ThermoFisher Scientific, Cat. No. {"type":"entrez-nucleotide","attrs":{"text":"W11261","term_id":"1285566","term_text":"W11261"}}W11261, 1:100 dilution) to label myofiber membranes. Next, slides were washed with PBS and subsequently cover slipped with Vectashield Hardmount containing DAPI (Vector Laboratories, Cat. No. H-1500-10). Images were obtained at x20 magnification using an Evos FL2 Auto microscope (ThermoFisher Scientific) and tiled/merged images of the entire muscle section were used for analysis. The number of perfused and total capillaries density measurements were quantified on thresholded images using a particle counter in Fiji/ImageJ and the quantified results were expressed as percent perfused and total number per area of the muscle section. Skeletal myofiber cross-sectional area (CSA) was determined using MuscleJ (⁷), an automated analysis software developed in Fiji.