title: The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.
abstract: We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.
authors.affiliation: Department of Cell Pathology, United Medical School, University of London, St. Thomas's Hospital, United Kingdom.
authors.forename: H
authors.initials: H
authors.lastname: Shimizu
authors.forename: A
authors.initials: A
authors.lastname: Ishida-Yamamoto
authors.forename: R A
authors.initials: RA
authors.lastname: Eady
mesh_terms: D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000648:ultrastructure*; D001324:Autoantigens / Q000276:immunology; D001485:Basement Membrane / Q000276:immunology; D002352:Carrier Proteins; D003094:Collagen / Q000276:immunology; D003102:Colloids; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D006046:Gold; D007150:Immunohistochemistry; D007633:Keratins / Q000276:immunology; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000276:immunology; D012834:Silver; D012867:Skin / Q000276:immunology*; D000097805:Collagen Type XVII
chemical_list: D000906:Antibodies; D000941:Antigens; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003102:Colloids; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D006046:Gold; D007633:Keratins; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D012834:Silver; D000097805:Collagen Type XVII; C095667:Dst protein, mouse