Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas

Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates o f cell proliferation are;conducted prospectively. Therefore, the opportunit y exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU ep itope was masked by formalin vs methacarn fixation in the assessment of cel l proliferation. Mammary carcinomas from animals pulse-labeled with BrdU we re trisected. A portion was frozen and the remaining two portions were fixe d in 10% neutral buffered formalin or methacarn far 24 hr, processed, embed ded in paraffin, and sections stained for incorporated BrdU using a peroxid ase immunohistochemical staining technique. Antigen retrieval techniques al so were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in un fixed frozen sections (17.5%). Formalin fixation alone dramatically suppres sed the labeling index (0.3%), which was only partially recovered using var ious antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recomme nded for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.