Background

Semi-quantitative immunohistochemistry (IHC) has been widely used for investigating protein expression and localization within tissues (Matkowskyj et al., 2000; Cregger et al., 2006; Taylor and Levenson, 2006; Braun et al., 2013; Fedchenko and Reifenrath, 2014; Bauman et al., 2016; Pike et al., 2017; Lu et al., 2018; Crowe et al., 2019), and various software and methods have been used in semi-quantiative IHC to conduct deconvolution and downstream analysis. Some methods require advanced coding/mathematical experience or the use of subscription-based software that may not be feasible for every scientist (Matkowskyj et al., 2000; Shu et al., 2016; Guirado et al., 2018). Fiji (ImageJ) is a freely available software (version 1.2; WS Rasband, National Institute of Health, Bethesda, MD). Publications using ImageJ for image quantification are often directed towards the creation of specific plugins for ImageJ or lack a step-by-step protocol (Varghese et al., 2014; Chen Y, 2017; Guirado et al., 2018). However, there is no integrated protocol that includes a detailed but simple procedure of conducting such steps, including the use of ImageJ software. There are many open online forums where users post their questions about using Fiji ImageJ for image quantification purposes. This is often due to publications not providing sufficiently detailed methods sections, which makes it difficult for the reader to reproduce the data. It is also time-consuming for researchers who are not initially familiar with ImageJ software to figure out how to use this software. The current protocol provides an example for conducting semi-quantitative analysis of IHC images using ImageJ.