Autoradiography of water-soluble cell components

The method used depends largely on whether precise localisation is required or whether a certain amount of diffusion of the soluble compounds can be tolerated or is required. In the latter case rapid air drying (at 37°C) of cells labelled with tritiated thymidine allows thymidine phosphates to diffuse a little way to form a halo around the nucleus (Adams, 1969a). For precise localisation it is necessary to freeze the cells in isopentane or freon held at liquid nitrogen temperature and then to subject them to lyophilisation. This drying 

Rather than being lyophilised cells may be freeze substituted (Pearse, 1953). After treating cells with isopentane at liquid nitrogen temperature they are flooded in several changes of absolute methanol at the temperature of solid C02 for 2-3 h. 

Once again if a certain amount of diffusion can be tolerated the fixed slides may be dipped into liquid emulsion and quickly dried in a horizontal position (Adams, 1969a). Drying vertically leads to a stream of grains trailing away from cells as the soluble radioactive compounds are washed out by the liquid emulsion. 

To avoid the problems of unswollen stripping film and to reduce diffusion to a minimum, partly dried liquid emulsion can be added to fixed slides (Miller et al., 1964a Finbow and Pitts, personal communication). 

Incorporation of bromodeoxyuridine is enhanced by incubating in the presence of fluorodeoxyuridine (to block endogenous synthesis of thymidine — 11.8.2) and access of the antibody to the fixed cells is ensured by partial denaturation or brief nuclease treatment (Goncharoff et al., 1986). After development of the peroxidase reaction cells can be counterstained, for example, with neutral red. 

---