Start by incubating the rat skeletal muscle fibers with TMRE at room temperature for 20 minutes. Next, launch the confocal microscope standard software, and choose the configuration framework. Under the Hardware Configuration dialogue box, select Laser, and check the Helium Neon 543 option.
Access the acquisition dialogue box via the Acquire framework, and select the XYZ panel. Then, select the 512x512 format with a 400 hertz speed. Under the displayed pinhole panel, select arbitrary units, and input three area for the pinhole.
In the Bean Path settings dialogue box, select a 20 x water immersion objective with a 0.7 numerical aperture, and an emission wavelength window of 576 to 700 nanometers. Choose DD 488543, and set the laser power for the 543 laser at 15%After a 20-minute incubation, change the incubation medium twice. Next, prepare the recording chamber with a 0.15 millimeter boro silicate glass cover slip.
Transfer the fiber bundles to the chamber containing 200 microliters of relaxed solution. Utilize the confocal microscope's bright field mode to identify viable fibers for the fluorescent recording of mitochondria. Adjust, gain, and offset by scanning the fluorescence using the Live button.
Choose low fluorescent intensity values for a background of nearly zero arbitrary units and a mitochondrial signal between 100 and 200 arbitrary units. Select a pixel size between 150 to 190 nanometers, and adjust the zoom factor in the XY dialogue box to acquire the fiber's full width. In the Z stack dialogue box, set the Z distance starting at a fiber depth of 15 micrometers up to 22 micrometers, and choose a Z step size of 3 micrometers.
Click the Start button to acquire the confocal images, and obtain a Z stack composed of three confocal images acquired at 15, 18, and 21 micrometers of depth. The confocal images of an exercised lean wrap muscle fiber show an expected record of skeletal muscle fiber mitochondria with a consistent fiber pattern. In contrast, the obese rat muscle fiber shows substantial alterations in mitochondrial content and distribution.
