Current methods of assessing immunohistochemistry center on semiquantitative visual grading scales. More objective methods utilizing digital quantification offer superior precision and, presumably, higher confidence with image comparison. However, their cost often remains prohibitive, and there is little customizability to separate subsections of interest in the tissue. Here we describe a method using two open-source software programs to analyze the intensity and density of signals in immunohistochemistry-stained tissue sections that account for tissue heterogeneity and allow for direct comparison between two samples. This method allows for quantitative assessment of epidermal protein expression. We herein demonstrate this workflow using an epidermal stain tothymic stromal lymphopoietin.
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