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