Immunostaining of keratin and vimentin in epidermis: comparison of different post-embedding immunogold techniques for electron microscopy.
The present study compares different post-embedding staining methods, including conventional and low-temperature embedding techniques, for demonstration of the keratin and vimentin cytoskeleton of epidermal cells, applying commercially available polyclonal and monoclonal antibodies. Immunogold staining (5-nm particles) was performed on the following material: (a) osmium-fixed and Durcupan-embedded material, etched with various solutions; (b) aldehyde-fixed material embedded in Lowicryl K4M at 4 degrees C and -35 degrees C; (c) aldehyde-fixed material embedded in Lowicryl K11M at -60 degrees C with and without cryoprotection with glycerol. In conventionally embedded material we failed to demonstrate intermediate filaments, whereas they were stained after low-temperature embedding with Lowicryl, i.e., K4M 4 degrees C, K4M -35 degrees C, and K11M -60 degrees C. The keratin and vimentin cytoskeleton reacted exclusively with polyclonal antibodies. The best results for antigenicity as well as structural preservation were achieved by post-embedding staining of K4M -35 degrees C-embedded material. Antibodies to keratin stained the cytoskeleton in keratinocytes of all epidermal layers. Filaments were decorated in a linear alignment. Antibodies to vimentin stained the cytoskeleton of Langerhans cells and melanocytes. In these cells a linear distribution pattern of the reaction product along the filaments and an extrafilamentous cluster formation were observed, indicating staining of vimentin and a vimentin-associated protein.
Mahrle
G
G
Department of Dermatology, University of Köln, West Germany.
Schulze
H J
HJ
Kuhn
A
A
Wevers
A
A
D000911:Antibodies, Monoclonal; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D014746:Vimentin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014746:Vimentin; D007633:Keratins