Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

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Abstract
Bromodeoxyuridine (BrdUrd) immunohisto-chemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0:05% proteinase at 37°C for 20 min and hydrolysis with 1 N HCl at 37°C for 20 min was sufficient to detect BrdUrd immunore-activity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd, immunohistochemistry in cell-kinetic studies.