Heat-induced antigen retrieval restores electrostatic forces: prolonging the antibody incubation as an alternative.

The term antigen retrieval was introduced by Shi et al. (1) to describe a process of heating formalin-fixed paraffin-embedded tissue sections for improved immunoreactivity of tissue antigens with their specific antibodies. Although it has currently become an essential part of immunohistochemistry, the exact nature of this process remains unknown. The following report will describe the rationale and results of experiments that associate the restoration of electrostatic (coulombic) forces with the functional basis of antigen retrieval. Critical support for this was derived by the application of relevant tenets regarding the sensitivity of immune reactions to pH and ionic strength and through the use of prolonged antibody incubations. For the majority of the investigated tissue antigens, prolonging the antibody incubation time from a standard 10 minutes to 60 minutes represented an effective alternative to heat-induced antigen retrieval. The report will carefully weigh the advantages and disadvantages of prolonged antibody incubations versus antigen retrieval procedures.