Heat-Assisted Stretching of Paraffin Sections on Hot Plate Weakens Immunoreactivity of Orotate Phosphoribosyltransferase

Abstract

Orotate phosphoribosyltransferase (OPRT) is the key enzyme for the phosphorylation of 5-fluorouracil (5-FU), the rate-limiting step for acquiring its anti-tumor effect. Since high enzyme activities and mRNA levels of OPRT are said to be associated with 5-FU chemosensitivity of cancer cells, the immunohistochemical demonstration of OPRT can be expected to contribute to the selection of patients who suffer from 5-FU-sensitive cancer. During a study for establishing the appropriate immunostaining condition using rabbit antiserum in formalin-fixed, paraffin-embedded sections of human cancer, we unexpectedly uncovered the fact that heat-assisted stretching of paraffin sections on a hot plate just after sectioning was critical for preserving OPRT immunoreactivity; namely, stretching sections briefly at 70°C or higher significantly reduced the immunoreactivity. Overnight drying of the sections in an oven at 37°C or 60°C did not influence the immunoreactivity, but pretreatments, including 0.2% trypsin, 0.002% proteinase K, and pressure cooking in 10 mM citrate buffer, pH 6.0 and pH 7.0, and 1 mM ethylenediaminetetraacetic acid solution, pH 8.0, seriously deteriorated the OPRT epitopes. The immunoreactivity was relatively resistant to overfixation, though weakened after fixation in formalin for four weeks. Xenografts with high OPRT enzyme activities showed distinct positive cytoplasmic staining, but those with low OPRT activities were equivocal. OPRT expression in routinely processed cancer tissues also provided valuable data.