Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously.
We describe a reproducible method for combining tritiated thymidine ([H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [3H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [3H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified. The inhibition of uptake into DNA of [3H]TdR from 0.23 to 1.85 MBq (6.25 to 50 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [3H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.
Hume
W J
WJ
Department of Dental Surgery, Leeds University School of Dentistry.
Thompson
J
J
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D001973:Bromodeoxyuridine / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D004247:DNA; D001973:Bromodeoxyuridine; D013936:Thymidine