Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers.
For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, hence, labeling at electron microscope resolution does not add essential information. The scope of this paper is to compare five permeabilization methods for pre-embedding labelling for electron microscopy. We aim for a method that is easy to use and suitable for routine investigations. For our ongoing work, special attention is given to labeling of the cell nucleus. Accessibility of cytoplasmic and nuclear antigens is monitored with a set of different marker antibodies. From this investigation, we suggest that prefixation with formaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure before using a detergent (Triton X-100 or Brij 58) to permeabilize or remove the membranes. The experimental conditions for labeling should be checked first with fluorescence or fluorescence-gold markers by fluorescence microscopy. Then either ultrasmall gold particles (with or without fluorochrome) with silver enhancement or, if the ultrasmall gold particles are obstructed, peroxidase markers are advised. The most promising technique to localize scarce antigens with good contrast is the combination of a pre-embedding peroxidase/tyramide-FITC or -biotin labeling followed by an on-section colloidal gold detection.
Humbel
B M
BM
Department of Molecular Cell Biology, Institute for Biomembranes, Utrecht University, The Netherlands. bruno@accu.uu.nl
de Jong
M D
MD
Müller
W H
WH
Verkleij
A J
AJ
D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured / Q000648:ultrastructure; D003902:Detergents; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D017979:Gold Colloid; D006801:Humans; D007089:Image Enhancement; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence; D010539:Permeability; D009195:Peroxidase; D012503:Saponins; D013301:Streptolysins; D016610:Tissue Embedding; D016707:Tissue Fixation; D014439:Tyramine
D003902:Detergents; D005456:Fluorescent Dyes; D017979:Gold Colloid; D012503:Saponins; D013301:Streptolysins; D009195:Peroxidase; D014439:Tyramine