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Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers
B M Humbel1, M D de Jong, W H Müller
1Department of Molecular Cell Biology, Institute for Biomembranes, Utrecht University, The Netherlands. bruno@accu.uu.nl
Insights
This study compares five permeabilization methods for pre-embedding labeling in electron microscopy. Prefixation with formaldehyde/glutaraldehyde and detergent treatment are crucial for preserving ultrastructure and improving antigen accessibility for scarce antigens.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Immunolabeling
Background:
- On-section labeling is often unsuccessful for scarce or embedded antigens.
- Pre-embedding labeling methods can compromise ultrastructural details.
- Electron microscopy requires methods that preserve fine cellular structures.
Purpose of the Study:
- To compare five permeabilization methods for pre-embedding labeling in electron microscopy.
- To identify an easy-to-use method suitable for routine investigations, especially for nuclear antigens.
- To optimize techniques for localizing scarce antigens with high contrast.
Main Methods:
- Comparison of five permeabilization techniques for pre-embedding immunolabeling.
- Utilizing formaldehyde/glutaraldehyde prefixation followed by detergent treatment (Triton X-100 or Brij 58).
- Validation using marker antibodies for cytoplasmic and nuclear antigens, and fluorescence microscopy.
Main Results:
- Prefixation with formaldehyde/glutaraldehyde is essential for ultrastructural stabilization before detergent permeabilization.
- Pre-embedding labeling requires careful optimization, initially checked with fluorescence microscopy.
- Ultrasmall gold particles with silver enhancement or peroxidase markers are recommended when gold particles are obstructed.
Conclusions:
- A combination of pre-embedding peroxidase/tyramide-FITC or -biotin labeling followed by on-section colloidal gold detection offers high contrast for scarce antigens.
- Effective pre-embedding labeling for electron microscopy necessitates ultrastructural stabilization and optimized permeabilization.
- The study provides a framework for selecting and optimizing methods for challenging immunolabeling applications.
Abstract:
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.
