Snap Freezing of Muscle Tissue for Sectioning: A Protocol to Obtain Ultrathin Sections of Rapidly Frozen Murine Muscle Tissue

Snap freezing is a technique by which tissue specimens are rapidly frozen to ultracold temperatures. It helps retain the native structure of DNA, RNA, and proteins inside the tissue.

To snap freeze the limb muscle, begin by taking freshly extracted muscle tissue from the hindlimb of a mouse.

Next, take liquid nitrogen in a styrofoam box and immerse a beaker containing isopentane - a highly conductive liquid - into liquid nitrogen.

Freeze the isopentane till the solution turns viscous. Simultaneously, apply a drop of liquid freezing media on top of a cork piece.

Position the hindlimb muscle tissue by holding its tendon and embed the tissue piece vertically in the liquid freezing medium. This orientation helps in the subsequent determination of the cross-sectional area of the muscle.

Submerge the cork piece with attached muscle tissue briefly into the pre-chilled isopentane bath. This aids in the rapid and even freezing of muscle tissue.

The freezing medium solidifies at low temperature and forms a gelatinous layer around the muscle tissue, holding it in place. Place the muscle block in a cryo-microtome to obtain thinly sliced sections.

Place the sections on glass slides and store them at a lower temperature for further histological analysis.

When all of the muscles have been collected, immerse the bottom half of a plastic 50-milliliter beaker containing isopentane into liquid nitrogen. When the isopentane becomes slightly viscous with a solid white laminate lining visible inside the beaker, pour a few drops of embedding medium on a chuck.

Then, pick up a muscle by the tendon and carefully position the end of the fresh muscle on top of the embedding medium, holding it vertically relative to the cork. Then, keeping the muscle vertical, dip the chuck and attached muscle into the isopentane bath for 7 to 15 seconds until the tissue is completely frozen. A well-frozen specimen will appear chalky white. Then, place the muscle in a specimen tube for immediate storage at negative 80 degrees Celsius.

To preserve the RNAs and enzymatic structure and activity of the samples, it is required to freeze the muscle tissues as soon as possible after their extraction.

To section the frozen muscle tissue, first, set the working temperature of the cryostat inner chamber to negative 23 degrees Celsius. Allow the negative 80 degrees Celsius specimen to acclimate to the working temperature for a few hours. Then, obtain multiple 8 micron thick sections of the specimen at the mid-belly region of the muscle, perpendicular to the mounting axis, collecting the sections on room temperature glass slides as they are cut. Keep the samples inside the cryostat chamber until all of the sections have been collected. Then, store the samples at negative 80 degrees Celsius until further analysis.