Protease antigen recovery decreases the specificity of bromodeoxyuridine detection in formalin-fixed tissue

Abstract

Incorporation of halogenated nucleotide analogues is often used to assess DNA synthesis and to quantitate cellular proliferation. Multiple antibodies have been developed to bromodeoxyuridine (BrdUrd) and it is the most frequently utilized substrate. Because the immunodetection of incorporated BrdUrd requires DNA denaturation or nuclease digestion, most of these antibodies are not reactive in tissues or cells fixed with crosslinking agents. Antigen retrieval techniques utilizing protease digestion restore BrdUrd antigenicity and permit the detection of BrdUrd in formalin-fixed tissue. However, during the development of a double label immunohistochemical protocol to quantitate proliferating alveolar Type II cells, we noted nucleus-specific staining in lung sections from animals that had not received BrdUrd. Therefore, we systematically analyzed the specificity of the immunohistochemical detection of incorporated BrdUrd in formalin-fixed tissue after protease digestion. Enzymatic antigen recovery diminished the specificity of the BrdUrd reaction product and caused false-positive staining with the BU-1, B44, and BR3 monoclonal antibodies. Staining was less prominent with Bu20a but was more specific. Protease antigen recovery may decrease the specificity of BrdUrd immunodetection. Appropriate controls are required when enzymatic digestion is used to detect incorporated BrdUrd in formalin-fixed tissue. The type and duration of fixation, antibody to BrdUrd, protease, and tissue may affect the specificity of the staining pattern.