Fig. 1

MyoSight identification of fiber borders, fiber-type, peri- and central nuclei. Representative images illustrating fiber border identification, fiber-type recognition, and peri- and central nuclei counting in the soleus from a WT mouse. a Representative image of original laminin stain in soleus of a WT mouse. b The FIJI “Find Edges” tool is used to enhance weak laminin staining. c Gaussian blurs are used to connect the breaks in the laminin staining separating adjacent fibers. d Fiber segmentation lines overlayed on original laminin stain. e Segmentation lines are colored to match laminin stain, and the image is flattened to enhance the laminin stain. f The flattened image is thresholded. g Individual regions of interest are created for each fiber. h All channels are combined for manual corrections of fiber borders and fiber-type. Representative images of fibers stained with antibodies to i MHC I, j MHC IIa, k MHC IIb, and l merged MHC immunofluorescent staining for all fiber-types in the soleus of a WT mouse with fiber borders overlaid. Representative MHC I/MHC IIa hybrid fiber defined by average pixel brightness for a fiber exceeds the threshold in two channels. m MHC I, n MHC IIa, o MHC IIb, and p merged. MHC immunofluorescent staining for all fiber types in the soleus of a WT mouse with fiber borders overlaid. Arrows indicate a hybrid fiber. q Representative images of nuclei staining in the soleus of an mdx mouse with fiber borders overlaid. r Perinuclei counting. The nuclei stain is subjected to watershed segmentation with a cross placed over the centroid of each nuclear region. Arrows indicate nuclei whose centroid is inside the fiber border and counted as fiber specific perinuclei. s Central nuclei counting. The fiber regions are reduced in size to include only the central region of each fiber. Arrows indicate nuclei whose regions overlap with the central region of a fiber and counted as a centralized nuclei. t Representative images of all MHC immunofluorescent staining and nuclei staining with fiber borders overlaid. Arrows indicate peri- and central nuclei from panel r and panel s, respectively. Scale bars are 20 microns (a–h), 40 microns (i–p), and 20 microns (q–t)