Skeletal Muscle Histology/Immunofluorescence

In this section I will list a number of useful stains/enzymatic reactions and immunofluorescent techniques for skeletal muscle cryosections that I have used/modified over the years, some will be basic, others will be a bit more complicated.

Choice of Muscle

Just a quick discussion on the choice of muscle for analysis, as some muscles are better suited to specific stains. I tend to use three hindlimb muscles for histology, the extensor digitorum longus (EDL), the soleus (SOL) and the tibialis anterior (TA). The EDL and the TA muscles are contain predominantly fast-twitch, glycolytic, type II fibers, while the SOL muscle contains predominantly slow-twitch, oxidative, type I fibers (in rat, or a mix of type I and type II fibers in mouse). An experiment that uses both EDL and SOL muscles will allow you to compare changes in the response of  glycolytic vs oxidative fibers to your desired intervention (disease state, pharmaceutical, genetic modification etc.). Specific stains often lend themselves more to one muscle than the other, for example, a stain for type I fibers is (generally) more useful in the SOL than the EDL.

Sample Preparation for Cryosectioning – Fresh frozen technique

Before starting, prepare an insulated container with liquid nitrogen, and a 100ml pyrex jar with ~30ml isopentane. Carefully place the pyrex jar containing isopentane into the liquid nitrogen, so it is partially submerged (DO NOT allow liquid nitrogen and isopentane to mix). When you can see white ‘blooms’ appear on the bottom of the pyrex jar, the isopentane is beginning to freeze and is ready (avoid allowing the isopentane to freeze completely).

Skeletal muscles should be carefully removed from the hindlimb in line with institution approved protocols. For fresh-frozen samples, the muscle should be laid out straight and flat, it should not be twisted or stretched. I like to use the top of a plunger from a 1ml syringe with two sides cut-off to create a long-thin strip at the top of the plunger on which to lay out the muscle. Carefully cover the muscle in OCT (Optimal Cutting Temperature compound) or similar, taking care to prevent bubbles on the muscle. Place to OCT covered muscle (on the plunger) into the semi-frozen isopentane for 20-30 seconds. Immediately transfer the frozen muscle to a freezer tube and store at minus 80 degrees.

Sample Preparation for Cryosectioning – Whole tissue PFA fixation technique

This technique is useful if structural integrity is important, this comes at the expense of enzymatic activity and the ability to use leftover muscle for western blotting, RNA isolation or other analyses. I use this technique if I plan to examine co-localization using immunofluorescence.

Remove skeletal muscle/s of interest as described above, and tie to a toothpick using a small piece of suture or cotton, making sure that the muscle is tied at a length that is taught but not stretched (this helps to maintain the fibers at a ‘resting’ length). Place the muscle attached to the toothpick into a 15ml Falcon tube containing 0.1-0.5% ice-cold PFA diluted in PBS (use sufficient volume so the muscle is completely covered). Keep the muscle in PFA on ice for ~2hrs (larger muscles such as the TA may need longer), and then wash the muscle three times in ice-cold PBS. After washing, the muscle can be removed from the toothpick, and mounted in OCT as described above.

4 thoughts on “Skeletal Muscle Histology/Immunofluorescence

  1. Lim Chai Ling

    Hello James,

    Could you please provide with the immunocytochemistry staining procedure with Pax 7? I have experienced very high background all these while. Thank you so much.

  2. shruthi

    Hello James,
    I’m a research fellow in the Neurophysiology department. NIMHANS. Bangalore. India. I also work on skletal muscle in motor neurodegenerative disease(ALS). I have a doubt regarding the immunofluorescence staining of skeletal muscle. Isn’t it necessary to perfuse the animal before using the tissue for Immunohistochemisry?

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