Bx61 wide field microscope

PM injury following LC-induced injury was assessed as before (Vila et al., 2017 (link)) using PO dye, which is excluded from myofibers with intact sarcolemma. Immediately after the LC assay, muscles were trimmed of tendons, blotted, weighed, and then incubated while held at L_o in a 0.2% PO solution at room temperature for 30 min, washed in Ringer solution, and quickly frozen in isopentane prechilled with liquid nitrogen. Frozen cross sections of 10-µm thickness were cut, mounted with fluorescent mounting medium (DAKO), and viewed under a fluorescent microscope to identify the presence of PO. Fields, containing the majority of the muscle cross sections were photographed and scaled under identical conditions at ×10 magnification using Olympus BX61 widefield microscope. By thresholding for unstained muscle, we identified fibers that showed no uptake of PO from fibers that showed PO uptake. From each muscle cross section, the area occupied by PO-labeled fibers ranging from minimal to maximal dye uptake was measured and expressed as a percentage of the complete muscle section area. PO-labeled fibers at the edges of the sections were considered as artifact and were excluded from analysis.