Automatic nonsubjective estimation of antigen content visualized by immunohistochemistry using color deconvolution.

We describe a method for the automatic, nonsubjective estimation of 3,3' diaminobenzidine (DAB) in digital images obtained from routine central nervous system immunohistochemistry using freely available, platform-independent public domain image processing software. This technique estimates the amount of antigen visualized but does not measure antigen content directly. Combined with whole brain section high-resolution scanning, a "virtual dissection" (extracting the region of interest) makes it possible to estimate relative antigen content in either subcellular structures, specific brain regions, or in whole tissue sections at magnifications up to 40×. The digital image is processed using Ruifrok and Johnston's color deconvolution method to separate the brown DAB chromogen from the hematoxylin counterstain on a microscope slide. A monochrome image representing the DAB content is then subjected to frequency analysis using NIH-ImageJ and a weighting calculation to estimate the amount of DAB (antigen) as a dimensionless index. The method described produces results that agree with enzyme-linked immunosorbent assays, and is automatic and nonsubjective. The method could easily be adapted to other types of tissue or cell cultures.