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

• Published in: Bio-protocol Vol. 9; no. 24
• Main Authors: Crowe, Alexandra R, Yue, Wei
• Format: Journal Article
• Language: English
• Published: United States Bio-protocol LLC 20.12.2019
• Subjects: ImageJ Fiji; Immunohistochemistry; DAB staining; Hematoxylin; OATP1B1; Semi-Quantification
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/bioprotoc.3465

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.