High-pressure cryoimmobilization of murine skin reveals novel structural features and prevents extraction artifacts.

Cryoimmobilization by high-pressure freezing (HPF) and subsequent freeze substitution has been proven as an effective method to preserve tissues. Here, we demonstrate for the first time that a comprehensive morphological and ultrastructural preservation of mouse skin throughout all its layers can be achieved in this way. Using conditions limiting tissue-extraction during freeze substitution, we could prevent the massive interdigitation of cell membranes, the loss of tubular structures of the Golgi complex, the aggregation of keratin to electron-dense bundles, the formation of round-shaped keratohyalin aggregates, the dispersion of locally organized ribosomes, the excessive aggregation of material at hemidesmosomal plaques, the massive extraction of material from the basement membrane and the adjacent dermal region, and the dissociation of components of the dermal matrix. Taken together, HPF in combination with freeze substitution emerges as a highly sensitive tool for morphological and ultrastructural analysis.