The overall goal of this protocol is to identify adult skeletal muscle satellite cells via immunofluorescence. This method can answer key questions related to the number and distribution of muscle stem cells in both physiological as well as pathological conditions. The main advantage of the technique is that one can identify muscle stem cells in their physiological environment with only limited manipulation.
Demonstrating the procedure will be Faiza Naz, a postdoc from my laboratory. To begin, prepare a liquid nitrogen bath by pouring liquid nitrogen into a Dewar flask. Next, pour 10 milliliters of methylbutane into a 20-milliliter plastic beaker and transfer the beaker to the liquid nitrogen bath.
Lay the dissected tibialis anterior muscle onto the flat surface of a small plastic syringe plunger. Next, cover the dissected muscle with a few drops of Optimal Cutting Temperature compound or O.C.T.Remove the beaker of methylbutane from the liquid nitrogen bath, then thaw the surface using the handles of a pair of scissors. Quickly dip the O.C.T.covered tissue into the melted methylbutane to snapfreeze it.
Next, use forceps to detach the embedded tissue from the plunger and transfer it to a 1.5-milliliter microcentrifuge tube. Then, store the microcentrifuge tube in the liquid nitrogen. To start cryostat sectioning, add a drop of O.C.T.compound onto the center of the specimen holder of a cryostat and allow it to partially solidify.
Then, stick the embedded tissue into the O.C.T.compound with the ankle side down and knee side up. Next, mount the specimen holder onto the cryostat and cut the tissue in 10-micrometer intervals. Collect four to eight cryosections on each frost-coated slide.
Dry the sections at room temperature for three hours, then collect the slides in a slide box and store them at minus 80 degrees Celsius. Next, remove a slide from the slide box and warm it to room temperature. Then, use a liquid blocker pen to mark an area around the cryosections to prevent liquids from flowing off the side.
In a fume hood, add 300 to 500 microliters of 4%paraformaldehyde to the marked area. Incubate the slide at room temperature for 10 minutes to fix the sections. Next, rinse the paraformaldehyde from the slide with PBS in a slide container.
To begin antigen retrieval, fill a slide container with 200 milliliters of citrate buffer and transfer the slide to the container. Then, transfer the slide container to a pressure cooker at high pressure for 10 minutes. The antigen retrieval step is optional if the sections are dried at room temperature for 30 minutes and proceed immediately.
Then the antigen retrieval step is not necessary. But if the sections are dried at room temperature for more than three hours then stored at minus 80 degrees Celsius, the antigen retrieval step is required. Next, cool the slide container under running water for 10 minutes.
Then transfer the slide to a new container with 200 milliliters of PBST and rinse with PBST. Add water-soaked lab wipes to the tray, then remark the rinsed slide with a PAP pen and place it in the tray. Add 200 microliters of blocking solution to the slide.
Then cover the tray and incubate at room temperature for 30 minutes. The blocking step is very important because the Pax7 antibody being used is a mouse mono-clonal antibody. The mouse-on-mouse blocking step reduces the unspecific bindings.
Then, rinse the slide with PBST in the slide container and return it to the tray. Add 200 microliters of Primary Antibody mix to the slide and incubate at room temperature for one hour. Next, rinse the slide with PBST, then add 200 microliters of Secondary Antibody mix and incubate in the dark at room temperature for one hour.
After the incubation period, rinse the slide with PBST. Dilute DAPI in a slide container and incubate the slide in the container for three minutes. Then, rinse the DAPI with PBST for five minutes.
Finally, mount the slide with mounting media and apply a coverslip. In this study, satellite cells are successfully visualized in adult resting muscle sections under a fluorescent microscope. The background noise usually seen with adult muscle tissue and certain types of fibers is reduced by using mouse-on-mouse blocking.
The specific immunostain signals are enhanced by using secondary antibodies conjugated with bright Alexa dyes. A two-photon confocal microscope captures a sharper image compared to the wide-field fluorescent microscope. The same protocol can also be applied to muscle tissue from juvenile mice, mouse embryos, and injured adult muscle tissues.
Both juvenile and embryonic muscle tissues depict more activated satellite cells or Pax7 myogenic precursors with relatively larger nuclei. Thus it becomes easier to visualize the satellite cells of younger mice than of adult mice. In the injured muscle tissue, more activated Pax7-positive cells are visible.
Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it is important to remember that noise coming from autofluorescence and unspecific mouse-on-mouse immuno-binding cannot be totally eliminated, but with this protocol the noise can be largely reduced. After watching this video, you should have a good understanding of how to identify satellite cells in muscle cryosections.
