Effects of glutaraldehyde and osmium tetroxide on newborn rat epidermal keratin

The effects of glutaraldehyde and osmium tetroxide (OsO4) fixation on ultrastructural and biochemical changes of epidermal keratin were investigated. Morphometrically, the apparent size of keratohyalin granules was smallest when granular cells were fixed with OsO4, whereas those fixed with glutaraldehyde were larger, and those double-fixed with glutaraldehyde and OsO4 were largest. The ultrastructure of tonofilaments appeared different in each type of fixation. Biochemically, cross-linking glutaraldehyde with purified keratin from cornified cells formed a high molecular weight product with a blue shift of the absorption maximum and an increase in absorbance. Detection of the absorption spectra of OsO4- keratin reactions revealed that a new absorption maximum around 410 nm developed in glutaraldehyde-treated keratin, while one around 315 nm appeared in nontreated keratin. In addition, glutaraldehyde pretreatment increased by 40% the initial reaction rate and by 23% the total amount of OsO4 reduction. Sodium dodecyl sulfate gel electrophoresis demonstrated that degradation of keratin molecules occurred in both glutaraldehyde-treated and untreated keratin after 2 hr of exposure to OsO4. These findings indicate that keratin molecules are chemically modified by fixatives routinely used for electron microscopy. We consider that this chemical information plays an essential role in the appropriate interpretation of the ultrastructure of epidermal cells.