A Method for 14C and 3H Double-Label Autoradiography

Abstract

The present study describes and validates a 3H/14C double-label autoradiographic method in which separation of the labels was obtained by sequential film exposures to film types sensitive to 14C only and to both 3H and 14C, respectively. The error in assuming a pure 14C image on the first film was 2–3%. A linear subtraction equation was developed for calculation of the 3H activity expressed in 14C equivalents as the difference in tissue activity between the second and the first film exposure. The actual 3H activity in the tissue could be obtained by multiplying the result by 25 (14C to 3H conversion factor). The subtraction procedure was validated for absolute 14C and 3H activities of 100–1,300 nCi/g and 600–11,000 nCi/g, respectively, and for relative 3H/14C activities between 5 and 10. Self-absorption of 3H in white matter was corrected for by multiplication by 1.61 (self-absorption coefficient). This factor was close to unity for 14C.