The overall goal of this procedure is to freeze skeletal muscle for optimal microscopic morphology. This is accomplished by first dividing the specimens to account for all of the desired tissue uses. In the second step, the muscle tissue is mounted in such a manner that the orientation is preserved while protecting the tissue from excess moisture.
Next, the mounted specimen is frozen in a freezing isop pentane bath. In the final step, the specimen is stored in a manner that prevents thawing and refreezing. Ultimately, the quality of the preservation can be evaluated by histological evaluation of the frozen muscle tissue.
Generally, individuals new to this method will struggle because very minor differences in freezing protocols can lead to significant artifacts in microscopic skeletal muscle morphology. Demonstrating this procedure today is Dr.Hui Meg, who's my primary assistant in both my research and clinical laboratories, and she'll be assisted by Ms.Jennifer Tinklenberg. No.Begin by subdividing the isolated skeletal muscle into fragments appropriate for the desired studies for the pieces of tissue for electron microscopy.
Cut and about 0.5 by 0.2 by 0.2 centimeter strip of muscle from the sample with a longitudinal axis of the sample parallel to the longitudinal axis of the original muscle to minimize the handling and any confusion during em processing. Then place a fragment of muscle no greater than two millimeters at its thinnest dimension directly into the desired electron microscopy fixative after fixation, place the muscle in the electron microscopy fixative buffer, and then send the tissue to an electron microscopy core facility for processing for histological, biochemical, or molecular studies. Include as much of the muscle as possible to minimize the sampling bias while keeping the samples less than 1.5 centimeters in size.
To avoid any freezing artifacts, treat all of the muscles from the same study similarly, to prevent any artifactual differences in the fiber size due to differences in handling. Next, thoroughly blot each specimen with a paper towel to remove the excess moisture until the tissue adheres slightly to the towel. Then place only enough adhesive such as OCT onto a base such as this piece of cork to provide a foundation for the oriented muscle.
Carefully place the muscle into OCT with the majority of the muscle protruding from the OCT so that the tissue to be sectioned is not in contact with the OCT. Now, add isop pentane to a metal cup until it reaches a depth of approximately three to four centimeters. Put on thermal safety gloves and remove the lid of the liquid nitrogen tank.
The most critical aspects of this are waiting until the isop pentane is adequately cold before trying to freeze the muscle, and also freezing and storing the muscle in a way that prevents thawing. Place the metal cup in contact with the liquid nitrogen so that the level of liquid nitrogen on the outside of the cup is above the level of the isop pentane on the inside of the cup. Observe the isop pentane to detect the formation of solid white pebbles of frozen isop pentane on the bottom of the cup indicating that the liquid has reached the appropriate temperature venues pre chilled forceps to freeze the specimen in the Isop pentane for approximately 10 to 20 seconds.
Finally, place the frozen specimen into a pre-cool container and keep it on dry ice until transfer to a minus 80 degree Celsius freezer. In this first image, appropriately frozen skeletal muscle showing a tight apposition of the myo fibers to the surrounding tissue and a clearly visible cytoplasmic compartment can be observed. The formation of freezing artifacts from excessive moisture is most commonly encountered in muscle tissue that has been previously immersed in saline and insufficiently dried, or in muscle tissue, has been in direct contact with OCT mounting media at the site of sectioning.
In contrast, these images of tissue frozen only in a minus 80 degree Celsius freezer or liquid nitrogen provide examples of freezing artifacts that are produced by suboptimal temperatures or the speed of freezing respectively, while the avoidance of freezing artifacts provides optimal histological results and improperly frozen tissue can be thawed and then refrozen under tightly controlled conditions to allow the redistribution of the water within the fibers as evidenced by the tissue in this image. The degree of morphological preservation in the rozen specimen can be excellent. After watching this video, you should have a good understanding of how to prepare skeletal muscle specimens for pathological studies requiring frozen muscle.
