High-throughput Analysis of Capillary Density in Skeletal Muscle Cross Sections

Capillary density in skeletal muscles is key to estimate exercise capacity in healthy individuals, athletes, and those with muscle-related pathologies. Here, we present a step-by-step, high-throughput semi-automated method for quantifying capillary density from whole human skeletal muscle cross-sections, in areas of the muscle occupied by myofibers. We provide a detailed protocol for immunofluorescence staining, image acquisition, processing, and quantification. Image processing is performed in ImageJ, and data analysis is conducted in R. The provided protocol allows high-throughput quantification of capillary density.Key features• This protocol builds upon the method and results described in Abbassi-Daloii et al. (2023b).• It includes step-by-step details on image acquisition and image processing of the entire muscle section.• It enables high-throughput and semi-automated image quantification of capillary density.• It provides a robust analysis for determining capillary density over the entire muscle cross section.Graphical overview