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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
Computer-aided image analysis applied to immunogold-silver staining: evaluation of proliferating cell nuclear antigen
1Department of Oral Pathology, Meikai University School of Dentistry, Saitama, Japan.
Insights
This study demonstrates that combining immunogold-silver staining (IGSS) with computer-aided image analysis enables precise antigen detection in tissue samples. This method offers a significant advantage for quantitative assessment of immunostained sections, improving diagnostic accuracy.
Area of Science:
- Biomedical Imaging
- Immunohistochemistry
- Digital Pathology
Background:
- Immunohistochemistry is crucial for diagnosing diseases like oral squamous cell carcinoma.
- Quantitative analysis of immunostained sections is challenging due to staining heterogeneity.
- Existing methods may lack the precision for detailed cellular analysis.
Purpose of the Study:
- To assess the feasibility of combining immunogold-silver staining (IGSS) with computer-aided image analysis.
- To evaluate the effectiveness of this combined method for detecting antigens in paraffin-embedded tissues.
- To determine if this approach enhances the quantitative assessment of immunostained sections.
Main Methods:
- Low-temperature IGSS was used to stain human oral squamous cell carcinoma tissue with PC 10 antibody against proliferating cell nuclear antigen (PCNA).
- ACAS 570 interactive laser cytometry and its software (COMPLEMENT DATA, DEFINE CELL, MARK AREA) were employed for analysis.
- Quantitative image analysis was performed on selected cells, measuring integrated value, area, perimeter, and shape factor.
Main Results:
- PCNA-positive cells showed dark nuclear staining with heterogeneous intensity.
- A 'complemental image' replicating immunohisto-morphology was generated using a conventional light source and software.
- Quantitative data on cellular features were successfully obtained and presented in histogram form.
Conclusions:
- The combination of IGSS and computer-aided image analysis is feasible for antigen detection.
- This integrated approach provides a significant advantage for the quantitative assessment of immunostained sections.
- The method allows for detailed analysis of cellular morphology and antigen expression patterns.
Abstract:
Feasibility of the combination of the immunogold-silver staining method (IGSS) and computer-aided image analysis was assessed for the detection of antigen in an immunostained, paraffin-embedded section. Using low-temperature IGSS, we stained a specimen of human oral squamous cell carcinoma with a monoclonal antibody, PC 10, against a proliferating cell nuclear antigen (PCNA/cyclin), and the section was analyzed by ACAS 570 interactive laser cytometry. The PCNA-positive cells, exhibiting a heteromorphic texture, were contrasted by the dark staining of their nuclei, but showed heterogeneity in staining intensity from cell to cell. Using a conventional microscope light source rather than a laser, and by employing the COMPLEMENT DATA program (which permits inversion of the data values) installed in the ACAS 570 software system, we were able to obtain a 'complemental image' which replicated the real immunohisto-morphology. Approximately 30-35 cells from three different areas in the same section were selected by DEFINE CELL and MARK AREA programs, and quantitative image analysis was performed in terms of cell integrated value, area, perimeter, and shape factor indicated in histogram form. The combined utilization of IGSS with computer-aided image analysis was demonstrated to offer a crucial advantage for the quantitative assessment of immunostained sections.
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