Background

Preserving tissue integrity is paramount for comprehensive morphological studies. Previous methods employed for tissue preservation, including formaldehyde fixation and cryopreservation, have demonstrated both advantages and challenges [1]. Formaldehyde fixation, while stabilizing tissues through protein crosslinking, may alter the natural conformation of cellular structures and impede antigen accessibility during immunohistochemical analysis. Cryopreservation, involving rapid freezing in liquid nitrogen without the need for fixation, is a widely used method for long-term tissue storage. However, the sudden temperature drop associated with liquid nitrogen often distorts the morphology of skeletal muscles. The liquid nitrogen evaporates immediately as it comes into contact with the warm tissue. This vapor becomes trapped between the mold surface and the liquid nitrogen, essentially creating an insulated barrier preventing freezing [2]. This process creates artifacts where intracellular water crystallizes immediately, resulting in the formation of huge crystals that rupture the muscle fibers. To address this, isopentane is utilized as a medium to facilitate controlled freezing [3].

A beaker filled with isopentane is placed in liquid nitrogen such that one-third of the isopentane is submerged. The isopentane is considered good for cryopreservation when a frozen layer forms at the bottom. At this stage, the bottom of the tissue mold is submerged in the chilled liquid. The duration of tissue submersion varies depending on the size of the tissue. Larger tissue requires longer freezing times than smaller tissues. The freezing times for small tissues like the diaphragm or extensor digitorum longus (EDL) is 6–12 s [3]. However, mice have even smaller tissues (e.g., soleus) that would require a freezing time of less than 6 s. Hence, small skeletal muscle tissues remain susceptible to freeze-fractures due to their higher surface-to-volume ratio. This method of freezing is highly inconsistent due to the many variables, i.e., liquid-to-frozen phase change, temperature fluctuations, visual assessment of phase state, tissue size, submerged depth of the mold, and batch-to-batch variability. In this study, rather than visual assessment, we monitor the temperature of isopentane when used for cryopreservation and present a reliable and reproducible method of preserving mouse skeletal muscles for both large and small tissues.