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

Simultaneous detection of two different antigens on paraffin-embedded and f rozen tissues can be accomplished by double immunohistochemistry. However, many double chromogen systems suffer from signal overlap, precluding defini te signal quantification. To separate and quantitatively analyze the differ ent chromogens, we imported images into a Macintosh computer using a CCD ca mera attached to a diagnostic microscope and used Photoshop software for th e recognition, selection, and separation of colors. We show here that Photo shop-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 provid es superior results compared to color separation using bandpass filters. Qu antification of the individual chromogens is then provided by Photoshop usi ng the Histogram command, which supplies information on the luminosity (cor responding to gray levels of black-and-white images) and on the number of p ixels as a measure of spatial distribution.