Effects of Tissue Fixation on the Inhibition of Erythrocyte Pseudoperoxidase Activity in Immunoperoxidase Methods

Abstract

Incomplete inhibition of erythrocyte pseudoperoxidase activity during immunoperoxidase techniques can cause falsepositive results and produce problems in data interpretation. Here, we examined the relationship between tissue fixation method and the frequency of false-positive results. Mouse brain tissues were immersed in 10% neutral-buffered formalin, 10% formalin, or 100% ethanol for 1 d, 3 d, 1 week, or 2 weeks. After treatment with hydrogen peroxide to block endogenous peroxidase activity, paraffin-embedded sections were immunostained for glial fibrillary acidic protein. False-positive results were observed when sections were fixed with 10% formalin or 100% ethanol for 1 d to 2 weeks but not when sections were fixed with 10% neutral-buffered formalin for durations of 1 week or less. Moreover, the intensity of false-positive results was positively associated with increased duration of fixation. Together, these results indicate that the incidence of false-positive results depends on the type of fixative and the duration of fixation. Fixation with 10% neutral-buffered formalin within 1 week may help to minimize these false-positive results. We also examined the efficacy of Kardasewitsch's method for inactivating pseudoperoxidase activity. This method, however, does not seem to be applicable to immunohistochemistry (The J Histotechnol 31(4):165–168, 2008).Submitted July 14, 2008; accepted with revisions July 26, 2008