High-pressure freezing provides new information on human epidermis: Simultaneous protein antigen and lamellar lipid structure preservation. Study on human epidermis by cryoimmobilization

Current transmission electron microscopy techniques do not permit simultane ous visualization of skin ultrastructure and stratum corneum extracellular lipids. We developed a new procedure, which entails application of high-pre ssure freezing followed by freeze-substitution with acetone containing uran yl acetate, followed by low temperature embedding in HM20. Electrospray ion ization mass spectrometry showed that the amount of lipids lost during prep aration was minimal. The ultrastructure of cryoprocessed skin was compared with that of conventionally prepared skin samples. Cryoprocessing, but not conventional processing, enabled visualization of lipid stacks within epide rmal lamellar bodies, as well as the extracellular lipid domains of the str atum corneum and the ultrastructure within keratinocytes. Anti-filaggrin im munocytochemistry also showed, e.g., excellent preservation of filaggrin on cryoprocessed samples. Additionally, the cytosol of keratinocytes appeared to be organized in ''microdomain''-like areas. Finally, the stratum corneu m appeared more compact with smaller intercellular spaces and hence tighter cell-cell interactions, after cryoprocessing, than after conventional tiss ue preparation for transmission electron microscopy. We conclude here that only cryoprocessing preserves skin in a close to native state.