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. 2019 Dec 20;9(24):e3465.
doi: 10.21769/BioProtoc.3465.

Semi-quantitative Determination of Protein Expression using Immunohistochemistry Staining and Analysis: An Integrated Protocol

Alexandra R Crowe  1 , Wei Yue  1
Affiliations

Semi-quantitative Determination of Protein Expression using Immunohistochemistry Staining and Analysis: An Integrated Protocol

Alexandra R Crowe et al. Bio Protoc. .

Abstract

Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed procedure of how to measure or compare protein expression. Publications that use semi-quantification methods to quantify protein expression often don't provide enough details in their methods section, which makes it difficult for the reader to reproduce their data. The current protocol for the first time provides a detailed, step-by-step instruction of conducting semi-quantitative analysis of IHC images using ImageJ Fiji software so that researchers would be able to follow this single protocol to conduct their research. The protocol uses semi-quantitative IHC of organic anion transporting polypeptide (OATP1B1) as an example, and gives detailed steps on how to deconvolute IHC images stained with hematoxylin and 3, 3 - diaminobenzidine (DAB) and how to quantify their expression using ImageJ Fiji. The protocol includes clear steps for a reader so that this method can be applied to many different proteins. We anticipate this method will provide a practical guidance to the reader and make semi-quantification of proteins an easier task to include in publications.

Keywords: DAB staining; Hematoxylin; ImageJ Fiji; Immunohistochemistry; OATP1B1; Semi-Quantification.

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Conflict of interest statement

Competing interests No competing financial interests for this study.

Figures

Figure 1.
Figure 1.. Color Deconvolution Window.
The Color Deconvolution Window will be used to separate the staining of the IHC image. The H DAB vector separates the IHC image into DAB staining (brown staining) for the protein of interest and Hematoxylin (H) staining for the nucleus.
Figure 2.
Figure 2.. Deconvolution of IHC image.
Separation of the IHC image into hematoxylin staining for the nuclei (Color 1) and DAB staining for OATP1B1 protein expression (Color 2) in hepatocytes within human liver tissue. Color 3 panel is for another staining, if applicable. For DAB and hematoxylin staining alone, the Color 3 panel can be eliminated. In DAB and hematoxylin staining, as in current studies, a warning “X” is shown so that users understand that the amount of antibody staining ( i.e. , DAB staining) in this case cannot be mathematically quantified, as it is not stoichiometric.
Figure 3.
Figure 3.. IHC image pre-threshold.
IHC image converted to black and white pixels prior to thresholding the image (A). The threshold pop up window pre-threshold indicates the baseline threshold values (B).
Figure 4.
Figure 4.. IHC image post-threshold.
The background for the DAB staining in the IHC image was removed by adjusting the maximum threshold value (A). The minimum and maximum threshold values were set and applied (B).
Figure 5.
Figure 5.. Measurement of DAB staining.
The Set Measurements window to choose the options for output of each IHC image is shown. The suggested options for output are highlighted with red boxes (A). The Results output window is shown in (B), and contains the name of the image (Label), area of the image (Area), and mean grey value intensity (Mean).
Figure 6.
Figure 6.. Measurement of nuclei size.
The raw IHC image stained with DAB for OATP1B1 staining and hematoxylin for nuclei staining is used to measure the size of the nuclei. Using the straight-line tool, a line is drawn over the nucleus (A, white arrow pointing to yellow line). The distance of the line is measured by going to Analyze > Measure in the ImageJ Fiji toolbox panel. The Length (highlighted in red box) represents the diameter of the nucleus (B).
Figure 7.
Figure 7.. Quantification of nuclei in IHC image.
To measure the number of nuclei in each IHC image, the Analyze Particle window pops up after selecting Analyze > Analyze Particles (A). The size of the particle measured is set to the average diameter of nuclei to infinity. The options to summarize and exclude on the edges are selected prior to clicking okay. The summarize option leads to the summarized output of the count, total area, and the average size of the nuclei particles in the IHC image (B). Exclude on the eges indicates that no nuclei particles will be included in the analysis.
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