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Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy
I Cornelese-ten Velde1, J Wiegant, H J Tanke
1Department of Cytochemistry and Cytometry, Sylvius Laboratories, University of Leiden, The Netherlands.
Histochemistry
|January 1, 1989
Summary
Reflection contrast microscopy (RCM) offers enhanced sensitivity for detecting diaminobenzidine (DABox) in immunocytochemistry. This technique enables quantitative analysis of peroxidase, proving valuable for techniques like in situ hybridization (ISH).
Area of Science:
- Biomedical Imaging
- Cell Biology
- Immunohistochemistry
Background:
- Immunocytochemistry requires more sensitive and quantitative detection methods.
- Diaminobenzidine (DABox) precipitation is key in immunoperoxidase staining.
- Reflection contrast microscopy (RCM) offers potential for enhanced detection.
Purpose of the Study:
- To apply RCM to optimize immunocytochemistry sensitivity.
- To establish quantitative relationships between peroxidase and DABox.
- To validate RCM conditions using in situ hybridization (ISH).
Main Methods:
- Utilized a simple model system with RCM.
- Tested methods for increased sensitivity (imidazole, nickel, cobalt ions).
- Investigated enzyme reaction parameters and incubation times.
- Employed in situ hybridization (ISH) as a biological model.
Main Results:
- Imidazole addition showed the highest sensitivity.
- Nickel and cobalt ions enhanced color intensity.
- Quantitative relationship observed between peroxidase and DABox reflection.
- High avPO concentrations yielded the best contrast in ISH.
Conclusions:
- RCM is a sensitive and quantitative tool for immunoperoxidase staining.
- Optimized conditions enhance DAB staining sensitivity and specificity.
- RCM is feasible for quantitative immunoperoxidase studies.