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Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy
S Hussain1, I Fredriksen2, H Ringsevjen1
1Division of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Science, University of Oslo, Oslo, Norway.
Journal of Neuroscience Methods
|February 1, 2019
Summary
Researchers developed new antibodies by immunizing rabbits with glutaraldehyde-fixed antigens. These antibodies show improved sensitivity for detecting fixed proteins in electron microscopy, enhancing immunogold labeling of tissues.
Area of Science:
- Immunology
- Neuroscience
- Microscopy
Background:
- Antibodies are vital research tools, but their effectiveness is reduced by tissue fixation methods.
- Glutaraldehyde (GA) fixation, common in electron microscopy (EM), alters epitopes and hinders antibody-antigen interactions.
- This leads to inefficient labeling and low signal intensity in immunolabeling techniques.
Purpose of the Study:
- To develop antibodies with enhanced affinity for glutaraldehyde-fixed antigens.
- To overcome limitations in antibody-based detection of fixed biological samples.
- To improve sensitivity in post-embedding immunogold electron microscopy.
Main Methods:
- Immunizing rabbits with glutaraldehyde-fixed antigens (SNARE proteins SNAP-25 and VAMP2).
- Screening antibody performance using western blotting (WB).
- Performing quantitative post-embedding EM and light microscopy (LM) on fixed tissues.
Main Results:
- In-house antibodies bound more strongly to GA-fixed proteins than non-fixed proteins, unlike standard antibodies.
- Quantitative EM showed higher labeling intensities with GA-fixed antigens using the new antibodies.
- Both LM and EM demonstrated superior labeling of GA-fixed tissues compared to formaldehyde-fixed tissues.
Conclusions:
- Raising antibodies against GA-treated antigens improves antibody sensitivity for post-embedding immunogold EM.
- This approach enhances the detection of antigens in glutaraldehyde-fixed biological samples.
- The developed antibodies offer a valuable tool for high-sensitivity immunolabeling in EM research.
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