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

Protokół

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Muscle Freezing and Mounting

1. Immerse the bottom half of a small plastic beaker (50 mL) containing isopentane into liquid nitrogen. The isopentane is ready to use when it becomes slightly viscous and forms a solid white laminate lining the inside of the beaker (temperature: -160 °C).
   NOTE: Always use non-sparking bronze or aluminum hand tools. Avoid breathing product vapor. Use with adequate ventilation. If dealing with a spill and ventilation is impossible or impractical, wear a suitable respirator.
2. Freeze some embedding medium on a chuck (cork) by dipping it briefly (10 s) into isopentane.
3. Handle the muscle (e.g., the tibialis or gastrocnemius) by holding it by the tendon, vertically relative to the cork, in order to maintain the orientation of the fibers and to allow for cross sections.
4. Carefully position the end portion of the fresh muscle on top of the frozen embedding medium.
   NOTE: The muscle will stick. It is important NOT to completely surround the muscle with embedding medium. It is also important to maintain the vertical orientation for subsequent determination of the cross-sectional area.
5. Dip the chuck with the attached muscle into the isopentane bath (the usual freezing time is 7-15 s, depending on the specimen size and composition).
   NOTE: Immersion in the freezing solution should not last more than is needed to completely freeze the specimen. Freezing too long will fracture the tissue block, while too short a time will cause ice crystal formation. A well-frozen specimen will be chalky white.
6. After freezing the specimen, place it into a small plastic bag or specimen tube (50 mL) and immediately store it in a deep freezer at -80 °C or in liquid nitrogen.

2. Muscle Sectioning

1. Set the working temperature of the cryostat inner chamber to around -23 °C.
2. Allow the specimen (previously stored at -80 °C) to acclimatize at the working temperature (a couple of hours should be enough).
3. Cut multiple 8 μm thick sections of the specimen (preferably the tibialis anterior or gastrocnemius muscles) and collect them on glass slides.
   NOTE: Cut the section at the mid-belly region of the muscle. Also, for the sake of accuracy, cut the sections perpendicularly to the mounting axis.
4. Keep the glass slides inside the cryostat chamber. Do not let them thaw if the aim is to perform IF/IHC studies or enzymatic staining.
5. Store the slides at -80 °C for further analyses.

Materiały

Name	Company	Catalog Number	Comments
OCT	Tissue-Tek	4583
Cryostat	Leica	CM1850
Cork disks	Electron Microscopy Sciences	63305
Superfrost plus glass slides	VWR	48311-703