Fig. 1

Automated analysis pipeline using our macro using Fiji. Automated analysis of immunofluorescence-stained muscle sections using our developed macro in the Fiji platform. (1) The first step consists of image enhancement by eliminating background noise and increasing the contrast. (2) The macro includes a line of command to fill the gaps within the fibers and a list of user-modifiable exclusion parameters to improve fiber detection and reduction of artifacts. (3) Then, individual fibers’ borders are identified based on the dystrophin/spectrin staining. (4) Among the detected fibers, those positive for MyHC are identified and segmented as type II myofibers. (5) Unlabeled type I myofibers are then calculated by difference. Fibers’ morphology parameters are computed, and the results automatically saved as .xls files. The program automatically saves the segmentation results of all and each fiber type as a flattened image, as well as the color maps for the area and minor diameter size distribution