Abstract
BackgroundThe 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 FindingsIn 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/SignificanceAll 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.
Highlights
Cellular functions are determined by genome-wide gene expression, co-localization, and interactions of multiple proteins
Protein co-localization is typically conducted on thicker cryosections with immunofluorescence histochemical staining, and can be visualized at high resolution with a confocal microscope
We developed a new method to completely strip previous primary antibody to ensure multi-target chromogenic immunohistochemical analysis on the same paraffin section
Summary
Cellular functions are determined by genome-wide gene expression, co-localization, and interactions of multiple proteins. The development of multi-target chromogenic immunohistochemistry partially alleviated the problem [1] It still requires primary antibodies coming from different species. We describe a sequential method for chromogenic immunohistochemistry to study protein co-localization and differential expression on paraffin sections. The images from different antibody staining can be superimposed to visualize protein co-localization as well as differential expression across an entire tissue section. 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. Chromogenic immunohistochemical staining, in general, is not suitable for the detection of protein co-localization. We developed a new protocol, based on chromogenic immunohistochemical stain, for system-wide detection of protein co-localization and differential expression
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