Our research centers around skeletal muscle, which is comprised of cells with distinctive metabolic and contractile properties, characterize as slow or fast twitch fibers. We delve into the factors contributing to these differences and explore their implications in the context of aging, exercise, and various diseases. Measuring cell responses in different skeletal muscle fibers is often overlooked or not possible due to the examination of muscle homogenous.
The MyDoBID technique allows us to repeatedly analyze fiber type protein differences that were previously impossible to measure. This protocol accurately and efficiently identifies type one and type two muscle fibers from freeze-dried human skeletal muscle samples. This newly revised method provides a straightforward approach for researchers to investigate protein differences in skeletal muscle at the cellular level.
Notably, it facilitates the identification of type two X fibers, and these have historically been challenging to distinguish. The technique allows researchers to shed light on all fiber types, including the elusive type two X fiber. With the MyDoBID technique, numerous questions can now be answered.
For example, one can discover the properties of type two X muscle fibers and determine if there's evidence indicating preferential atrophy of type two X muscle fibers during aging or disease. One can also observe how a specific exercise intervention impacts each fiber type. To begin, position the freeze-dried muscle sample under the stereomicroscope and view it at low magnification.
With one pair of fine tissue dissecting forceps, hold the muscle in place and separate the small bundles of fibers with the other forceps. Isolate one bundle of fibers and gently tease them apart until single fiber segments are extracted from the bundle. Then shift the fiber to a clear area of the Petri dish.
Investigate each single fiber segment under 50X magnification. Attempt to further separate the fiber at one end. If the fiber begins to break, it is a single fiber.
Carefully collect the fiber with a pair of forceps and place it into the previously prepared aliquot of denaturing buffer. Tap the tube firmly on the bench three times. Afterward, vortex the fiber samples and briefly centrifuge them for five seconds at 2, 500 x g to ensure the sample settles at the bottom of the tube, let the samples rest at room temperature for one hour before storing them at minus 80 degrees Celsius for future use.
To start, prepare the four sheets of filter paper each measuring 12.5 by 7.5 centimeters. Stack two sheets together and place the stack in transfer buffer to soak through. Place the PVDF membrane in 95%ethanol and agitate the membrane on a rocker for one minute.
Recollect the ethanol, immerse the membrane in transfer buffer, and agitate for two minutes. Next, lay the soaking filter paper stack on a flat movable surface, such as a large lid, and flatten it using a roller. Using a gel releaser or tweezers, carefully position the PVDF membrane on the stack.
Afterward, place a single sheet of dry filter paper on this membrane and glide the roller over the paper to absorb any excess buffer that's on the membrane surface. Lift off the top filter paper without rubbing against the membrane. Take isolated fiber samples from the freezer and thaw them at room temperature before centrifuging and thoroughly suspending the sample.
Place a one microliter droplet of each sample in the center of the designated membrane area. Avoid touching the membrane with the pipette tip. Allow the sample droplets to absorb entirely into the membrane for 15 minutes.
Then using a gel releaser or tweezers, carefully lift the membrane from the damped filter paper stack. Place it onto a dry sheet of filter paper and leave it for at least five minutes to dehydrate. Reactivate the membrane after the sample spots have turned completely white.
Then proceed with immunolabeling the target protein. Learn how to use the signal panel intensity to classify the intensity of the MHC isoforms and actin signals from the dot blot images. Strong, medium, and little target protein detection is shown by saturated, moderate, and faint signals, respectively.
For fiber type identification, initially compare the myosin heavy chain IIa and myosin heavy chain I results. Document the fibers that display only a single myosin heavy chain isoform, with either saturated or moderate signal intensity. Record the fibers detected with actin and no detection of myosin heavy chain I or IIa as a potential type two X.If a faint myosin heavy chain isoform signal is present with a moderate desaturated actin signal, recorded as unidentified.
Discard samples with faint or undetected target proteins. Fibers displaying saturated or moderate signals of both MHCIIa and MHCI should not be used for fiber type specific preparation. After MyDoBID, prepare a type one and two samples by combining fibers with saturated or moderate signals for the respective MHC isoform.
Use 10 microliters of each fiber type-specific sample and separate them on a precast gel by SDS Page following the manufacturer's guidelines. Use a gel imager to detect the target MHC isoform following the manufacturer's protocol. Compare the signal intensity of each MHC isoform in all fiber type-specific samples.
Fiber type ID is confirmed by the correct MHC isoform at moderate or saturated signal intensity. Immunolabeling of the dot blot allowed the identification of type two fibers, type one fibers, and potential type two X fibers. The fiber type specific samples were validated using Western blotting and myosin heavy chain-specific antibodies.
