Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

The overall goal of this procedure is to avoid pitfalls when preparing frozen sections from muscle biopsies, such as creating freezing artifacts. This is accomplished by first freezing the muscle rapidly in two methyl butane chilled on liquid nitrogen. After the muscle biopsies equilibrate in the cryostat, they are embedded in optimum cutting temperature, medium at room temperature in small increments with quick freezing using aerosol cooling and heat extraction.

Next seven micron sections are sliced and transferred to warm, positively charged glass slides. The final step is to stain the tissue sections as needed to evaluate the tissue's histology. Ultimately correctly prepared muscle biopsies result in well-preserved tissue histology without freezing artifacts.

So the main advantage of this technique over other existing methods, such as paraffin embedded tissue sectioning, is that it's very quick and it does not alter endogenous antigen structure or function. This method can help answer key questions in the health and disease of muscle biology, such as growth, regeneration, inflammation, fibrosis, and necrosis. The implication of this technique extend towards therapy or diagnosis of muscle disorders because it allows determination of condition of healthy and diseased muscle.

Though this method can provide insights into different forms of muscular dystrophy, it can also be applied to other systems such as cachexia associated with terminal stages of cancer and aids and loss of muscles in elderly populations. Begin with euthanizing a mouse with an overdose of isof fluorine under a fume hood and in a secondary level of containment, such as a bell jar. Confirm the animal's death by firmly squeezing its footpad.

Now, wet the fur with 70%alcohol. This prevents loose hair strands from sticking to the muscles. Proceed by peeling the skin off the limb using a number five fine forceps.

Carefully separate the muscle of interest from the bone starting from the tendon. In this case, the tibias anterior is isolated. After excising the muscle, cover it in OCT and lay it flat on a labeled disposable freezing mold.

More OCT will be added later before slicing. Ensure that the muscle is in its normal physiological orientation along the head to tail axis and that the muscle isn't stretched. Now chill.

Some isop pentane in a steel beaker using liquid nitrogen. Stir it intermittently over three to five minutes with a plastic spatula. When a white precipitate forms the isop pentane is at its optimal temperature at this point, dip the loaded molds into the solution.

Small muscles such as the diaphragm and extensor digitorum longest requires six to 12 seconds. Larger muscles require 15 to 20 seconds over freezing can lead to cracking. The next step is to put the frozen tissues on dry ice to let the residual isop pentane evaporate, which takes about 15 to 20 minutes.

Once the tissue is dry, rapid in foil and store it in a minus 80 degrees Celsius freezer until it can be sectioned. Prior to sectioning. Transport the frozen tissues on dry ice to the cryostat.

Transfer the tissue to a cryostat chamber set between negative 24 and negative 20 degrees Celsius, and let them equilibrate for 30 minutes. The tissues will not fully thaw at this temperature. Next, embed the tissue first.

If available, turn on the Peltier Bay cooling. Second, apply a uniform thin layer of OCT on the specimen disc and let it cool. Third, once the OCT has frozen on the bottom but is still liquid on the top, insert the tissue, orient the tissue perpendicular to the OCT for transverse sections and parallel to the OCT for longitudinal sections.

Now use an aerosol cooling spray to quickly freeze the OCT. Then touch the uninvented part of the tissue with a heat extractor and hold it to the tissue for 45 seconds. Do not let the muscle thaw at any step during this process.

If the cryostat does not have peltier bay cooling, use aerosol cooling to quickly freeze the OCT around the tissue. Now, add another thin layer of OCT around the muscle spray with aerosol cooling spray and extract the heat as in the previous step. Keep adding OCT in small increments and quickly freeze the OCT using a combination of aerosol cooling spray and heat extractor until the whole muscle is covered.

Then put the heat extractor on the top of the tissue block and wait for five minutes before proceeding with sectioning. The specimen is now ready to mount on the specimen head. First, tighten the knob to secure the disc.

Make sure that the specimen disc is in good contact with the specimen head while the chamber gets to be between negative 24 and negative 20 degrees Celsius. Set the slice thickness to seven microns when the chamber is at temperature, start trimming the block. Use the course and fine adjustment to move the block.

When sections are ready to collect, press their top sides onto lamp warmed positively charged slides. If frozen correctly, the tissue face should be chalky white. However, a tissue that was frozen, improperly or thawed during the mounting process will appear red to pink.

The slides can be stored in the deep freezer for future analysis. The mid belly section of the tissue can be identified by measuring the cross section area from phase contrast images using the tracing function of an imaging software package. This analysis provides both area and the minimum ferret diameter measurements.

When these measurements begin to plateau, the mid belly has been reached. While manufacturers provide step-by-step instructions for most staining protocols, personal optimization may be required to achieve optimal staining intensities. The OCT does not need to be removed.

For staining, for optimal immuno staining, be sure to use a humidified chamber. It should be covered with perfil kept on a wet paper towel and sealed in a small box to avoid drying of sections. During the staining process for non immuno stained permanent mount slides, following the staining protocol, dehydrate the slides through increasing grades of alcohol.

Because mounting media is not missable with water, then clear the slides with a five minute bath in 100%xylene. This ensures that the slides do not become opaque over time. Then proceed with mounting and imaging sections of the mid belly region of the tibials anterior were stained.

Using hematin and eoin. Sections that are properly processed will have consistent morphology and even staining throughout improperly processed muscles that are freeze damaged or prematurely. Thawed appear to have multiple breaks in morphology and have the appearance of shredded wheat.

Immunohistochemistry was also performed on these sections. These stains were satisfactory. Fibronectin is stained in green and dappy is seen in blue.

After watching this video, you should have a thorough understanding of how to prepare a good frozen section from muscle biopsies for evaluation of muscle architecture. Once mastered, this technique can be done in approximately four to five hours if all these steps are done properly. While attempting this procedure, it is important to remember to prevent thawing of muscles Following this procedure.

Other methods like immuno staining and insight to hybridization can be performed in order to answer additional questions like growth and regeneration.