Detection of Bromodeoxyuridine in Paraffin- embedded Tissue Sections Using Microwave Antigen Retrieval is Dependent on the Mode of Tissue Fixation

bstract A simple routine microwave antigen retrieval procedure allows the sensitive detection of incorporated BrdU in pulse labeled cells. Of the two fixatives tested, Carnoy’s offers superior nuclear morphology, but with a sacrifice of immunostaining intensity. For investigations where animals are sacrificed within several hours after pulse labeling, Carnoy‘s fixative may prove adequate as a general fixative, but it is not known what effect it has on cellular antigens of interest. For our purposes, 10% neutral buffered formalin was found to be a superior fixative because of its ability to crosslink nuclear proteins and associated chromatin, resulting in more intense immunostaining for BrdU. In addition, we have found that formalin fixation coupled with microwave antigen retrieval is completely compatible with immunostaining of other antigens of interest. Introduction Bromodeoxyuridine (BrdU), an analog of thymidine, is incorporated into DNA during the S-phase of the cell cycle, and is a useful alternative to labeling proliferating cells with tritiated thymidine. Incorporated BrdU is detected immunologically with the use of monoclonal antibodies, and the resulting labeled cells can be analyzed either by flow cytometry, or microscopy. Since BrdU is detected immunologically, labeled cells can be observed within hours. In contrast, cells labeled with tritiated thymidine must be detected by autoradiography which can take weeks to months. Because BrdU is incorporated into proliferating cells, its use includes estimates of cell cycle kinetics, growth fractions, and the proliferative status of tissues. To study the proliferation potential of specific tissues, animals are injected with BrdU, sacrificed one or two hours later, and the tissues removed and fixed for microscopic analysis. A common fixative used for BrdU labeling is Carnoy’s, which is a mixture of acetic acid, alcohol, and chloroform [1]. To detect incorporated BrdU immunologically, the DNA must be denatured to allow antibody access. Denaturation of DNA in Carnoy’s fixed samples is usually accomplished by treatment with HCl. Stronger crosslinking fixatives such as formalin or glutaraldehyde can inhibit detection of BrdU, requiring a combination of enzymatic treatment with pepsin in addition to DNA denaturation with HC1 [1]. As an alternative for enzymatic digestion, microwave antigen retrieval can be used to unmask antigens of interest in tissues that have been fixed in formalin and embedded in paraffin [2], and this technique has been used successfully to detect BrdU in long-term labeled cells [3].