Detection of bromodeoxyuridine in formalin-fixed tissue. DNA denaturation following microwave or enzymatic digestion pretreatment is required

The present study was designed to assess the influence of antigen retrieval and/or DNA denaturation on the quantitative estimation of bromodeoxyuridin e (BrdU) in formalin-fixed paraffin-embedded tissue. Specimens of small int estine from rats injected with BrdU were routinely fixed and embedded in pa raffin. For antigen retrieval, sections were pretreated with microwave irra diation or enzymatically (pepsin or trypsin). Acid hydrolysis was used as a DNA denaturation method. Immunostaining of BrdU-labeled cells was performe d. The best results, regarding tissue morphology and immunostaining, were o btained with microwave pretreatment followed by acid hydrolysis. Enzymatic pretreatment resulted in damage of tissue morphology and/or high background staining. Microwave alone, without DNA denaturation, resulted in a lower p ercentage of BrdU positive cells. The significance of validation studies is emphasized when the level of positivity for a prognostic marker, such as B rdU, is assessed.