Validation of bromodeoxyuridine immunohistochemistry for localization of S-phase cells in decalcified tissues. A comparative study with tritiated thymidine autoradiography

Immunohistochemical detection of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdUrd), which is incorporated by S-phase cells, offers a convenient way of studying the proliferation kinetics of cells in normal skeletal tissues and in bone containing/derived tumours. To assess the validity of using this approach on decalcified, paraffin embedded tissues, the BrdUrd method was compared with tritiated thymidine (3H-TdR) autoradiography, using rat tibiae labelled with both 3H-TdR and BrdUrd, fixed in Carnoy's fluid and decalcified in EDTA, prior to routine paraffin embedding. The distribution of BrdUrd-labelled cells correlated with the sites of cell proliferation in the growing rat tibia. Independent studies with each method on paired serial sections of double-labelled tissue, showed a highly significant correlation (r = 0.81, p less than 0.0003) in the numbers of labelled cells seen in autoradiographs and immunostained sections from the proximal tibial growth plate. Combined BrdUrd immunohistochemistry and 3H-TdR autoradiography showed that the majority of labelled cells in cartilage, bone marrow, and fibrous perichondrium and periosteum had incorporated both labels. These results show that BrdUrd immunohistochemistry is a valid technique for the study of dividing cells in mineralized tissues after decalcification.