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Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy
Author(s) -
Van den Bergh B. A. I.,
Swartzendruber D. C.,
Bosvan der Geest A.,
Hoogstraate J. J.,
Schrijvers A. H. G. J.,
Boddé H. E.,
Junginger H. E.,
Bouwstra J. A.
Publication year - 1997
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1046/j.1365-2818.1997.2200779.x
Subject(s) - ultrastructure , transmission electron microscopy , electron microscope , protocol (science) , microscopy , transmission (telecommunications) , materials science , chemistry , nanotechnology , microbiology and biotechnology , computer science , physics , biology , pathology , optics , anatomy , medicine , telecommunications , alternative medicine
The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO 4 ) and ruthenium tetroxide (RuO 4 ) in combination with potassium ferrocyanide (K 4 Fe(CN) 6 ) and potassium ferricyanide (K 3 Fe(CN) 6 ). Instead of fixing skin with either OsO 4 or RuO 4 , these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K 4 Fe(CN) 6 and K 3 Fe(CN) 6 in combination with RuO 4 were examined. Furthermore, we compared two dehydration solvents, methanol and acetone. The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO 4 and RuO 4 with K 3 Fe(CN) 6 , i.e. with Fe in its highest oxidation state (Fe 3+ ). No differences were observed between dehydration in methanol and acetone.