The effect of diffusion on the immunolocalization of antigen

Diffusion prior to tissue fixation may result in immunohistochemical localization of antigen at sites in which it does not occur in vivo. The extent and speed with which this may occur have been investigated with several antigens of different molecular weights in tissues which were left unfixed for various lengths of time. There was clear evidence of diffusion when fixation was delayed for as little as 1 h, resulting in focal labelling of cells which did not contain antigen in rapidly fixed tissues. Falsely localized antigen was particularly evident adjacent to sites containing high antigen concentrations and might be expected in areas permeated by antigen-containing tissue fluid. These findings substantiate the view that diffusion of antigen should be considered as a cause of false positivity in any immunolocalization study.