Prevention of non-specific interactions of gold-labeled reagents on tissue sections.
The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling. We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling. We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.
Roth
J
J
Interdepartmental Electron Microscopy, Biocenter, University of Basle, Switzerland.
Taatjes
D J
DJ
Warhol
M J
MJ
D000818:Animals; D006046:Gold; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis*; D008854:Microscopy, Electron; D051381:Rats; D013205:Staphylococcal Protein A
D013205:Staphylococcal Protein A; D007633:Keratins; D006046:Gold