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Updated: Aug 8, 2026
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
Selective quantitative analysis of the intensity of immunohistochemical reactions
Insights
This study introduces a new densitometric method for measuring immunohistochemical reaction intensity using image analysis. The technique accurately quantifies subtle differences in antigen concentration, crucial for precise biological research.
Area of Science:
- Biomedical Imaging
- Cell Biology
- Immunohistochemistry
Background:
- Immunohistochemistry (IHC) is vital for visualizing antigens in tissues.
- Accurate quantification of IHC reaction intensity is challenging, especially for small differences.
- Existing methods may lack sensitivity for detecting subtle variations in antigen expression.
Purpose of the Study:
- To develop and validate a novel densitometric method for precise IHC reaction intensity measurement.
- To enable the detection of small differences in relative IHC reaction intensity.
- To optimize image analysis for quantitative IHC.
Main Methods:
- Utilized a Kontron VIDAS image analysis system program for densitometry.
- Employed avidin-biotin-peroxidase complex and diaminobenzidine-HCl/H2O2 for enzyme visualization.
- Developed shade correction and image processing techniques, including subtractive correction and reference image division.
Main Results:
- The new method successfully measured small differences in IHC reaction intensity on gerbil Purkinje cells.
- Standardized preparation (section thickness, IHC protocol, no counterstaining) is critical for accurate results.
- Feasible evaluation of small intensity differences was achieved through specific shade correction and normalization/thresholding methods.
- Detecting small antigen concentration differences required additional image processing beyond basic correction.
Conclusions:
- A novel, precise densitometric method for IHC intensity measurement has been established.
- The method, when combined with standardized protocols and advanced image processing, allows for sensitive detection of antigen variations.
- This technique enhances the quantitative capabilities of immunohistochemistry in biological and medical research.
Abstract:
The present study reports a new method for the densitometric measurement of the intensity of immunohistochemical reactions. This method is based on a programm for the Kontron VIDAS image analysis system and has been designed for the measurement of small differences in the relative intensity of immunohistochemical reactions. Immunohistochemistry was performed with the avidin-biotin-peroxidase complex and diaminobenzidine-HCl and H2O2 for enzyme visualization. Several methods for shade correction and image processing were elaborated. The study was carried out on gerbil Purkinje cells using monoclonal antibodies raised against calbindin D28k. Prerequesites of correct measurement were standardized preparation, i.e., identical thickness of the paraffin sections, identical performance of immunohistochemistry, and avoidance of any counterstaining. The evaluation of small intensity differences of immunohistochemical reactions was found to be feasible either by substractive shade correction and standardized normalization or by shade correction by division by a reference image and standardized thresholding. Small differences in antigen concentration were not detectable without additional image processing.
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