Demonstration of Intracellular Localization of Calmodulin Antagonist by Wet-Mounting Radioautography

The purpose of the present study is to develop a radioautographic technique for demonstrating the localization of calmodulin antagonist, W-7, in CHOK1 cells. The cultured cells were labeled with 10 μ/m1 tritiated W-7 for 1 hr and were divided into the following 3 groups: (1) they were fully rinsed in buffer solution for 1 hr and doubly fixed in glutaraldehyde and osmium tetroxide solution, (2) they were washed and divided into three subgroups, which were prefixed in glutaraldehyde, paraformaldehyde or osmium tetroxide solution respectively and postfixed in osmium tetroxide solution, (3) they were incubated in the medium without tritiated W-7 for 30 mm, 1 hr, 3 hr or 5 hr respectively and doubly fixed in glutaraldehyde and osmium tetroxide solution. All the specimens were dehydrated and embedded in Epon. Thick and thin sections were wet-mounted with radioautographic emulsion and were exposed from one to several months with both light and electron microscopic radioautography. Among the radioautograms fixed with various fixatives, glutaraldehyde showed the largest number of silver grains mainly localized in cytoplams of CHO-K1 cells. This approach to radioautography seems useful for ultrastructrual localization of calmodulin. W-7