Myofiber necrosis is characteristic of multiple neuromuscular disorders and plays a central role in pathogenesis. A specific, reliable method for assessing muscle degeneration is important for diagnosis and translational research. This extremely adaptable technique allows the labeling of multiple antigen as same time in the same section within that myofibers.
It is possible with vital dyes. H&E staining is an empirical methods that is not adaptable for reliable and reproducible conjugation. IgG uptake staining, however, is specific for necrotic muscle cells and applicable to human biopsies.
This technique can provide insight into all neuromuscular disorders characterized by myonecrosis. After removing the hair from the leg of interest place the four week old male mdx animal in the supine position and secure the paws to a cork board. Using precision scissors or forceps remove the leg skin from the foot to the knee to expose the entire length of the tibia and tibialis anterior.
Use the scissors to make an incision between the tibia and the tibialis anterior. Use forceps to carefully remove the epimysium layer from the surface of the muscle. At the ankle, use the forceps to isolate the tibialis anterior tendon from the other tendons and gently pull up on the tendon to isolate it from the tibia and the surrounding muscles.
When the tibialis anterior belly has entirely separated hold the tendon up so that only the proximal part of the tibialis anterior muscle is attached to the knee. And gently sever the proximal tendon as close as possible to the patella. Place the tibialis anterior on a piece of gauze lightly dampened with saline to avoid drying.
Partially dip a beaker containing 70 to 100 milliliters of isopentane into a container of liquid nitrogen until the isopentane at the bottom of the beaker becomes a white solid. Carefully pre-label the other side of the corks so that the biopsy can be properly identified after freezing, and add approximately 0.5 milliliters of tragacanth gum onto a 20 by 11 by 8 millimeter circular piece of cork. Use forceps to embed the tibialis anterior into the gum distal tendon side up.
Holding the muscle with its axis perpendicular to the cork surface. At least half of the tendon side of the tibialis anterior should remain uncovered by the gum. Then quickly dip the cork with the embedded muscle into the chilled, unfrozen isopentane for about two minutes to allow complete freezing.
Obtain sections of the frozen tissue, set the cryostat temperature to minus 25 degree Celsius and use optimal cutting temperature compound to fix the tissue onto the cryostat cutting block. Trim the sample until the muscle belly is reached. Obtain seven to 10 micrometer sections collecting at least two sections per glass slide placing the slides at room temperature as the sections are captured.
Allow the tissues to dry for at least 20 minutes at room temperature in a ventilated environment. Then store the section at minus 80 degree Celsius until they're staining. Immunolabel the tissues.
First, thaw the slides at room temperature for at least 15 minutes in the ventilated environment. Next, delineate the sections with a hydrophobic pen. Fix the tissues with 2%paraformaldehyde for 10 minutes in a humid chamber.
At the end of a fixation rinse the sections with two 5 minute washes in fresh PBS per wash. Block any non-specific binding with 10%goat serum diluted in PBS. After 1 hour at room temperature label the sections with a primary antibodies of interest, diluted in 5%goat serum for 2 hours at room temperature.
At the end of the incubation, rinse the slides with three 5 minute washes in PBS follow by labeling with the appropriate fluorophore conjugated secondary antibodies for 45 minutes at room temperature protected from light. At the end of the incubation, wash the slides with three 10 minute washes in PBS. Mount the section with a fluorescent mounting medium containing 100 nanograms per milliliter of DAPI and a cover slip.
Then dry the slides overnight at four degree Celsius before imaging. Tibialis anterior muscles from four week old mdx mice often display heterogeneous profiles including poorly affected areas in degenerating and regenerating areas together in the same cross-section. Unaffected to mildly affected muscle areas contain myofibers of a large and homogenous size and nuclei at a low density that are mainly located at the periphery or in between myofibers.
IgG positive myofibers are generally absent in such health or mildly affected areas while IgG immunoreactivity within the myofibers indicates myonecrosis. Newly formed myofibers are present at a small size at their early stages of regeneration progressively enlarging as the muscle progenitors fuse in contribute to the syncytial cell. During this process, the myonuclei remain at the center of the regenerating tissue with clusters of small, centrally nucleated myofibers indicating recently regenerated myofibers To minimize the risk of artifacts it's important to keep in the muscles straight when freezing the tissue and at the correct temperature.
Isopentane and PFA should always be handled with caution and under a fume hood.
