Superheating Using Pressure Cooking: Its Use and Application in Unmasking Antigens Embedded in Methyl Methacrylate

Abstract

Unmasking of antigens in formalin fixed, paraffin embedded tissue sections for immunohistochemical staining has recently been reported using superheating with the aid of a pressure cooker. We describe the use and application of this technology to semithin sections of tissue that have been embedded in methyl methacrylate, the plastic we routinely employ for high resolution light microscopy immunohistochemical studies. In particular, we have investigated the use of superheating for the detection of bcl-2, CD3, CD79a, and Ki-67, as these antigens had previously proved more difficult to demonstrate following other pretreatment procedures. Using a protocol of superheating in 10 mM citrate buffer (pH 6.0) for 3 min, we easily localized all the antigens, with superior immunocytochemical staining to that achieved with microwave antigen retrieval. However, CD3 was best demonstrated when pretreatment with trypsin at 37°C was used in addition to pressure cooking. We have also shown that the choice and concentration of the accelerators N-N dimethylaniline or N-N tetramethylaniline employed for polymerizing the plastic affected immunocytochemical staining. (The J Histotechnol 21:231–236, 1998)