Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis

Simultaneous detection of two different antigens on paraffin-embedded and frozen tissues can be accomplished by double immunohistochemistry. However, many double chromogen systems suffer from signal overlap, precluding definite signal quantification. To separate and quantitatively analyze the different chromogens, we imported images into a Macintosh computer using a CCD camera attached to a diagnostic microscope and used Photoshop software for the recognition, selection, and separation of colors. We show here that Photoshop-based image analysis allows complete separation of chromogens not only on the basis of their RGB spectral characteristics, but also on the basis of information concerning saturation, hue, and luminosity intrinsic to the digitized images. We demonstrate that Photoshop-based image analysis provides superior results compared to color separation using bandpass filters. Quantification of the individual chromogens is then provided by Photoshop using the Histogram command, which supplies information on the luminosity (corresponding to gray levels of black-and-white images) and on the number of pixels as a measure of spatial distribution.