Visualization

1. Film autoradiography: In an X-ray cassette, the slides must be held firmly against the film with even pressure on all slides. This can be accomplished by gluing the back of the slides to a piece of stiff paper or poster board. The choice depends on the depth available in the cassette. The film is inserted and exposed for a few days at -70°C, and then developed. It can be compared to the same slide stained and cover slipped after exposure, or to adjacent sections stained and cover slipped.

2. Emulsion autoradiography: The NTB-2 is diluted 1:1 before use. In the dark room, use a clean spatula to transfer the room temperature emulsion to a sterile screw-cap centrifuge tube. Warm in a water bath to 45°C until the emulsion melts. Approximately 15 mL of gelled emulsion melts to 7 mL. Mix with 7 mL of deionized H20 using a clean slide, then dip a trial slide, and check for an even coating and freedom from bubbles, indicating the emulsion is melted and well mixed. Dip each slide, wipe the reverse side, and then stand the slides on end. Leave them to dry in the dark (13). Turn off even the safe light. After they are dry, 1-2 h later, pack them into black boxes with silica gel and seal them shut with plastic tape. Expose at 4°C for a few days to 1 mo.

3. Develop the slides in Kodak D19 for 2 V2 min. Rinse for 30 s in an acid stop bath, and fix in 5% (v/v) Na thiosufate. This can be followed by staining the section in many of the usual histological stains. Hematoxylin eosin is the most common (see Chapter 48). Follow by dehydration in ethanol and cover slip with a xylene-based mounting media. Brain sections can also be stained in neutral red nissal stain for 4 min, rapidly dehydrated by incubation in each alcohol for 15 s, cleared in xylene, and cover slipped. The light-red stain provides excellent contrast to the black grains of the autoradiographic signal.

4. Autoradiographs can be examined with bright-field microscopy or dark field. The bright field allows you to see the stained section and the grains in the emulsion in the same field. The dark field allows visualization of the autoradiographic grains only, allowing a quick scan for label above background and facilitating a grain density comparison over tissues of different staining densities.

Was this article helpful?

0 0

Post a comment