DEVELOPMENT OF AN OPTIMAL PROTOCOL FOR THE ULTRASTRUCTURAL EXAMINATION OF SKIN BY TRANSMISSION ELECTRON-MICROSCOPY

The intercellular lipid bilayers of the stratum corneum provide the pe rmeability barrier of the skin, To perform an electron microscopical e xamination of the ultrastructure of these bilayers, ruthenium tetroxid e fixation is required, In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation proced ures, using glutaraldehyde (GA) and the postfixatives ruthenium red, o smium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination w ith potassium ferrocyanide (K4Fe(CN)(6)) and potassium ferricyanide (K 3Fe(CN)(6)). Instead of fixing skin with either OsO4 or RuO4, these tw o fixatives were combined in one protocol, In addition, the use of RuR ed was introduced and the influence of both K4Fe(CN)(6) and K3Fe(CN)(6 ) in combination with RuO4 were examined, Furthermore, we compared two dehydration solvents, methanol and acetone. The most satisfying resul ts were obtained when the skin was prefixed in GA and postfixed in OsO 4 and RuO4 with K3Fe(CN)(6), i.e. with Fe in its highest oxidation sta te (Fe3+), No differences were observed between dehydration in methano l and acetone.