Related Experiment Video
Updated: May 24, 2026
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
System-wide immunohistochemical analysis of protein co-localization
Minjung Kim1, Virawudh Soontornniyomkij, Baohu Ji
1Department of Psychiatry, University of California San Diego, La Jolla, California, United States of America.
Insights
A new chromogenic immunohistochemical staining protocol enables system-wide detection of protein co-localization and differential expression. This method visualizes multiple proteins across entire tissue sections, overcoming limitations of traditional techniques.
Area of Science:
- Neuroscience
- Histochemistry
- Molecular Biology
Background:
- Immunofluorescence is common for co-localized protein analysis but limited to small regions.
- Chromogenic immunohistochemistry is generally unsuitable for detecting protein co-localization.
- A novel protocol was developed for system-wide protein co-localization and differential expression analysis.
Purpose of the Study:
- To develop a novel chromogenic immunohistochemical staining protocol.
- To enable system-wide detection of protein co-localization and differential expression.
- To overcome the limitations of traditional methods in visualizing protein interactions within tissues.
Main Methods:
- Developed a protocol using a removable chromogenic stain and an efficient antibody stripping method.
- Enabled sequential immunostaining with different primary antibodies, irrespective of host species.
- Scanned sections after each staining step and superimposed images for co-localization analysis.
Main Results:
- Successfully examined differential expression and co-localization of glutamic acid decarboxylase67 (GAD67) and parvalbumin in mouse cortex.
- Demonstrated that all parvalbumin-containing neurons express GAD67 protein.
- Visualized GAD67-positive neurons lacking parvalbumin among thousands of other neurons.
Conclusions:
- The new method offers a global view of protein co-localization and differential expression across entire tissue sections.
- Repeatedly using the same tissue section allows for combined assessments of multiple proteins.
- This technique enhances the understanding of protein interactions and expression patterns in complex biological systems.
Background:
The analysis of co-localized protein expression in a tissue section is often conducted with immunofluorescence histochemical staining which is typically visualized in localized regions. On the other hand, chromogenic immunohistochemical staining, in general, is not suitable for the detection of protein co-localization. Here, we developed a new protocol, based on chromogenic immunohistochemical stain, for system-wide detection of protein co-localization and differential expression.
Methodology/Principal Findings:
In combination with a removable chromogenic stain, an efficient antibody stripping method was developed to enable sequential immunostaining with different primary antibodies regardless of antibody's host species. Sections were scanned after each staining, and the images were superimposed together for the detection of protein co-localization and differential expression. As a proof of principle, differential expression and co-localization of glutamic acid decarboxylase67 (GAD67) and parvalbumin proteins was examined in mouse cortex.
Conclusions/Significance:
All parvalbumin-containing neurons express GAD67 protein, and GAD67-positive neurons that do not express parvalbumin were readily visualized from thousands of other neurons across mouse cortex. The method provided a global view of protein co-localization as well as differential expression across an entire tissue section. Repeated use of the same section could combine assessments of co-localization and differential expression of multiple proteins.
Related Concept Videos
01:22Immunocytochemistry and Immunohistochemistry
These...
01:20Immunogold Electron Microscopy