Autoradiographic Differentiation of Free, Bound, Pure, and Impure Thymidine-3H

Intestinal tissue from animals injected with tritium-labeled thymidine were examined by two autoradiographic methods. Conventional autoradiograms using fixed, solvent-dehydrated, paraffin-embedded tissues, and wet mounted, were compared with an autoradiographic method designed for localizing diffusible substances utilizing dry-mounted, freeze-dried frozen sections. The autoradiograms prepared by conventional methods show more than 95% of the activity located over the nucleus while, autoradiograms of intestinal tissue from the same animal prepared by the freeze-dried method shows only 88% of the activity over the nucleus. Animals injected with impure thymidine-3H, containing self-radiolysis decomposition products due to storage, led to significant alteration in the autoradiographic pattern, particularly those prepared by the freeze-dried method which show only 52% of the activity over the nucleus. Thus, conventional treatment of tissue extracted free unincorporated thymidine, metabolic products of thymidine-3H, and impurities of thymidine-3H. However, autoradiograms prepared by the dry-mounted freeze-dried method retained all the label.