A reproducible technique combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine for double labelling studies of cell proliferation in paraffin sections of tissues.
A method is described to combine tritiated thymidine autoradiography with immunoperoxidase detection of bromodeoxyuridine on the same paraffin sections. It overcomes the varied technical artefacts we encountered when first attempting to combine these techniques and results in preparations with extremely low peroxidase and autoradiographic backgrounds. In particular, we find it is important to avoid the use of detergents during immunostaining, otherwise grain counts are reduced and autoradiograph exposures need to be greatly increased, and to avoid excessive peroxidase staining which makes it difficult to visualize silver grains in the overlying emulsion. The advantages of a method to remove emulsion films using acid-alcohol, allowing the same sections to be dipped twice with a long and a short autoradiographic exposure, are presented. The routine combination of high quality tritiated thymidine autoradiography with clean immunoperoxidase staining of bromodeoxyuridine-positive nuclei provides a new and powerful cell kinetic, double-labelling method to augment existing techniques e.g. by labelling the same cells undergoing DNA synthesis in successive cell cycles.
Department of Dental Surgery, Leeds University School of Dentistry, U.K.
W J
WJ
Hume
D000818:Animals; D001345:Autoradiography; D001973:Bromodeoxyuridine / Q000032:analysis*; D002455:Cell Division; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D010232:Paraffin; D013936:Thymidine; D014316:Tritium
D014316:Tritium; D010232:Paraffin; D001973:Bromodeoxyuridine; D013936:Thymidine