Cryopreservation Method for Preventing Freeze-Fracture of Small Muscle Samples

Histological techniques to study muscle are crucial for assessing skeletal muscle health. To preserve tissue morphology, samples are usually fixed in formaldehyde or cryopreserved immediately after excision from the body. Freezing samples in liquid nitrogen, using isopentane as a mediator for efficient cooling, preserves the tissue in its natural state. However, this method is highly susceptible to freeze-fracture artifacts, which alter or destroy tissue architecture. Isopentane is most commonly used in a semi-frozen/liquid state that is visually assessed by the experimenter, which can pose a challenge when freezing multiple tissues at a time or maintaining a consistent temperature. Furthermore, tissue size is also a confounding factor; depending on the size, freezing times can vary. In this study, we compare two different options for using isopentane while cryopreserving tissue. We also present an easy and reproducible method of freezing the soleus tissue of mice using frozen isopentane. This method decreased the occurrence of freeze-fractures by an order of magnitude, to ~4%, whereas the traditional method of cryopreservation resulted in ~56% freeze-fracturing. Key features • A uniform and highly reproducible protocol for freezing any tissue that is prone to freeze-fracture. • Removes the need to maintain a mixed state of isopentane. • Optimized cryopreservation method for the soleus muscle of mice. • Allows for prevention of peripheral freeze-fracture in tissue, which is the most susceptible region to freeze-fracture damage. Graphical overview.