Calibration of 14C-plastic standards for quantitative autoradiography with 33P.

Abstract

Probes labeled with 33P have potential for widespread use in in situ hybridization because they are better able to detect relatively scarce mRNAs compared with probes labeled with 35S, but the relatively short half-life of 33P is a disadvantage when it is used as a radioactivity standard for quantitative autoradiography. To determine if plastic sections containing 14C can be used as standards for quantitative autoradiography with 33P, we co-exposed 33P-labeled liver paste sections and 14C-plastic standards to Hyperfilm beta max. The autoradiographic response of Hyperfilm beta max to these isotopes was almost identical. Second-order polynomial equations obtained from analysis of film relative optical density and radioactivity permitted derivation of tissue-equivalent radioactivity from the film optical densities produced by the 14C standards for 1-14-day exposures. These results validate the use of plastic 14C standards for quantifying 33P used in contact film autoradiography.