Tritium autoradiography

Key words Mo-B alloy, Grain boundary segregation, Precipitates, Particle-tracking autoradiography (PTA), Tritium autoradiography (TA). 

The nonquantitative detection of radioactive emission often is required for special experimental conditions. Autoradiography, which is the exposure of photographic film to radioactive emissions, is a commonly used technique for locating radiotracers on thin-layer chromatographs, electrophoresis gels, tissue mounted on sHdes, whole-body animal sHces, and specialized membranes (13). After exposure to the radiolabeled emitters, dark or black spots or bands appear as the film develops. This technique is especially useful for tritium detection but is also widely used for P, P, and 1. 

Cellular autoradiography techniques using radioactive nucleic acid probes have several features in common with nucleic acid immunocytochemistry. The method is based on the hybridization of radioactive probes to cellular targets and the subsequent exposure of photographic emulsion, which, when developed, reveals blackened (exposed) silver grains close to the site of hybridiza-hon. Hence, cellular autoradiography techniques permit excellent specihcity and localizahon of the hybridized probe—to 1 qm when tritium is the label used in the autoradiography-based method (9). 

FIGURE 25-3 Visualization of bidirectional DNA replication. Replication of a circular chromosome produces a structure resembling the Greek letter theta (0). (a) Labeling with tritium (3H) shows that both strands are replicated at the same time (new strands shown in red). The electron micrographs illustrate the replication of a circular E. coli plasmid as visualized by autoradiography, (b) Addition of 3H for a... 

Before concluding this section, the rather special technique of autoradiography must be mentioned. This makes use of the fact that ionising radiations will blacken a photographic plate. The technique has been used most elegantly by Park (47—49) to study the distribution of a tritium labelled urea-formaldehyde resin finish in textile fibres. 

Many tritium labelled receptor ligands are commercially available. The advantages of the use of tritium as an isotope for the radiolabelling of receptor ligands are its long half-life and the relatively safety of use. The low energy of the (3 particle is beneficial for autoradiography,... 

Timmons (12) showed that the water in a never dried freshcut tree, when exchanged with a series of organic solvents could be replaced with radioactively tritium labeled MMA (hydrogen-3). After polymerization and the use of autoradiography the MMA was located in the cell wall structure. If the same wood was dried normally, and then treated in the usual manner with tritium labeled IWIA, the polymer was located exclusively in the capillaries and none was found in the cell walls. 

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

Liver cells are then exposed in vitro to the test compound and incubated with tritium-labeled thymidine for about 18 hours. At the end of the incubation, the cells are fixed on slides and prepared for autoradiography. For that the slides are first exposed to liquid photographic emulsion, air-dried and following a 7-day exposure in the dark, exposed to developing solution. 

Autoradiography is a technique for locating radioactive compounds within cells it can be conducted with light or electron microscopy. Living cells are first exposed to the radioactive precursor of some intracellular component. The labeled precursor is a compound with one or more hydrogen ( H) atoms replaced by the radioisotope tritium (3H) e.g., [3H]thymidine is a labeled precursor of DNA, and [3H]uridine is a labeled precursor of RNA (Chap. 7). Various tritiated amino acids are also available. The labeled precursors enter the cells and are incorporated into the appropriate macromolecules. The cells are then fixed, and the samples are embedded in a resin or wax and then sectioned into thin slices. 

Nock B, Johnson AE, Feder HH, McEwen BS. Tritium-sensitive film autoradiography of guinea pig brain a2-noradrenergic receptors. Brain Res 1985 336 148-152. 

Figure 11. Microscopic autoradiography of liver of a mouse treated with tritium labelled CPIA-cholesterol (X540) 5 weeks after single intravenous injection of ca. 30 mg/kg of 3-H-[2R]-CPIA-cholesterol ester. 3-H label is localized in Kupffer cells (arrow and giant cells (long arrows).

In autoradiography, the metal is typically charged with tritium-enriched hydrogen [91, 92]. The diffusible hydrogen is then removed by outgasing, and the... 

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