The overall goal of this immunofluorescent staining protocol is to monitor the autophagic process in muscle satellite cells during skeletal muscle regeneration. This method can help answer key questions in the tissue regeneration field about understanding the contribution of autophagy in deriving muscle regeneration and monitoring autophagy in satellite cells. The main advantage of this technique is that the autophagic process can be directly detected in satellite cells in tissue section from unperturbed and damaged muscles.
Demonstrating this procedure will be Francesco Castagnetti, a great student from my laboratory. To induce acute skeletal muscle injury, load an insulin syringe, equipped with a 30 gauge needle, with 20 microliters of sterile filtered 10 micromolar cardiotoxin stock. Inject the cardiotoxin directly into the left tibialis anterior muscle of an equal number of male and female two month old, approximately 20 gram, C57 black six mice.
To assess the autophagic flux, starting at 24 hours post injury, administer 50 milligrams per kilogram chloroquine every 24 hours for four days by intraperitoneal injection. Chloroquine treatment is critical as it leads to LC3 accumulation and detectability under active autophagic flux conditions. Five days after the injury, wet the animals with 70%ethanol.
And use scissors to make a three millimeter perpendicular incision in the dorsal skin of the mouse at the hip level. Pull the skin towards the tail to expose the underlying muscles. And identify the tibialis anterior muscle.
Use forceps to grasp the tibialis anterior muscle by the tendon. And carefully pull the muscle up toward the knee. Then pull the two distal tendons in opposite directions to separate the tibialis anterior muscle from the extensor digitorum longus muscle and cut the proximal tendon.
Use the forceps to remove the thin fascia covering the muscle, taking care not to damage the tissue. And use a paper towel to remove any excess moisture from the sample. Place the excised muscle into the center of a tissue mold, containing optimal cutting temperature compound.
And use pre-chilled forceps to carefully lower the mold into liquid nitrogen chilled isopentane for 20 to 30 seconds. Then store the frozen sample at negative 80 degrees celsius. After at least one night at negative 80 degrees celsius, set the cryostat between negative 15 to negative 23 degrees celsius and use a drop of optimal cutting temperature compound to attach the muscle sample to the cryostat chuck with the bulk of the tissue oriented at the top of the chuck.
Then obtain seven to eight micrometer thick sections, collecting three to four tissue slices per slide. After all of the section have been acquired, air dry the tissue samples for 10 to 15 minutes, followed by hematoxylin and eosin staining. Check the quality of the sections under an optical microscope at a 10X magnification.
And fix the samples with 4%paraformaldehyde in an incubation chamber. After 10 minutes, remove the fixative with four five to seven minute washes in PBS. Then use a hydrophobic pen to draw a barrier around the tissue sections.
And cover the section with 200 microliters of negative 20 degree celsius methanol for a five minute incubation at negative 20 degrees celsius. Methanol permeabilization is critical as it facilitates a good retention of the LC3 staining. Next, wash the section four times in PBS as just demonstrated.
And block the tissue samples with an unconjugated affinity purified anti-mouse IGG FAB fragment for at least 60 minutes at room temperature. At the end of the incubation, incubate the specimens in 100 microliters of primary antibody mix at four degrees celsius overnight. The next morning, wash the slides with 1%bovine serum albumin in PBS four times for 10 minutes per wash.
Then incubate the samples with the appropriate secondary antibody cocktail for 45 minutes at room temperature protected from light. At the end of the secondary antibody incubation, wash the slides four times in PBS for five to seven minutes per wash. Followed by labeling with the second primary antibody cocktail for one to two hours at room temperature protected from light.
Wash the sections in 1%BSA and PBS four times. Followed by incubation in the appropriate second antibody for 45 minutes at room temperature protected from light. Then wash the samples in PBS and label the tissues with 200 microliters of DAPI per slide for five minutes at room temperature protected from light.
After one last set of PBS washes, remove the excess PBS from the slides and add 10 microliters of glycerol in PBS onto the center of each slide. Cover each slide with a coverslip, taking care to avoid bubbles. Select the 63X objective on a four laser confocal microscope integrated with an image capture system and analytical software.
Center the fields of active regeneration on the first slide to allow manual focus of the regenerative areas. Then locate the myoD positive muscle satellite cells positioned within the laminin labeled myofibers. And obtain images within at least seven fields from at least three mice per experimental group.
H and E staining reveals that while unperturbed muscles displayed generally invariable myofiber sizes, injured muscles typically featured disrupted myofibers that differ in size and are abundant in inflammatory infiltrate. Further, the central nucleation of the myofibers, that is absent in unperturbed muscles, is a sign of new fiber formation in mice undergoing active regeneration. MyoD positive muscle satellite cells express LC3 and laminin staining allows the proper localization of muscle cells in the niche beneath the basal lamina.
While uninjured mice do not demonstrate any muscle satellite cell activation as evidenced by their lack of myoD positive cells and LC3 signal. Once mastered, this technique can be completed in two days if it is performed properly. While attempting this procedure, it is important to remember to always keep the tissue sections hydrated.
After its development, this technique paved the way for researching the field of tissue regeneration to explore the autophagic process during regular and paralogical skeletal muscle regeneration in mice and human biopsies. After watching this video, you should have a good understanding of how to identify LC3 in satellite cells during muscle regeneration.
