Nis elements d image analysis software

Cross sections (10 μm thick) from the midbelly of the muscles frozen in optimal cutting temperature compound were obtained with a cryostat and fixed in acetone for 10 min at −30°C. The sections were warmed to room temperature (RT) for 5 min and rehydrated with PBS for 15 min. The sections were then incubated in solution A (PBS containing 0.5% BSA and 0.5% Triton X-100) for 20 min and probed with the indicated primary antibodies (dissolved in solution A) for 1 hour at RT. After washing with PBS, the sections were incubated with the appropriate fluorophore-conjugated secondary antibodies (dissolved in solution A) for 1 hour at RT. When nuclei staining was needed, the sections were subsequently incubated with propidium iodine (20 μg/ml) for 5 min. After a final wash with PBS, fluorescent signals from each secondary antibody or propidium iodine were captured with a DS-QiMc camera on an 80i epifluorescence microscope (both from Nikon) at RT, and the resulting monochrome images were merged with NIS-Elements D image analysis software (Nikon). Investigators that were blinded to the sample identification then used NIS-Elements D to measure the total fiber number per muscle cross section, average fiber CSA, myonuclei per fiber, macrophage density, and myonuclear localization.