Notes

1. Paraformaldehyde is toxic, so the fixative should be prepared in a fume hood. It is very important to prepare correctly the fixative to obtain the best preservation of the tissue. Do not overheat the solution more than 60°C.

2. Use the sodium salt of PIPES, which is easier to dissolve than the free acid. 50X Denhardt's is 1% (w/v) BSA, 1% (v/v) Ficoll 400 and 1% (w/v) polyvinylpyrrolidone. 20% SDS is prepared in water and autoclaved. The NaCl, PIPES, EDTA, and Denhardt's solutions must be filtered through a 0.22-|im filter before adding to the hybridization buffer.

Dissolve the yeast tRNA in DEPC water. The ssDNA is dissolved in water by stirring in a beaker. It takes time to dissolve. Aliquots of both solutions are kept at -20°C.

Fig. 2. Emulsion autoradiography using a labeled RC3 riboprobe combined with immunohistochemistry for T3 receptor 02 isoform in the rat retrosplenial cortex. Photography was carried out using a 63X immersion objective. Note the black silver grains over receptor positive cells. The scale bar equals 25 pm. Data are from ref. 31.

Fig. 2. Emulsion autoradiography using a labeled RC3 riboprobe combined with immunohistochemistry for T3 receptor 02 isoform in the rat retrosplenial cortex. Photography was carried out using a 63X immersion objective. Note the black silver grains over receptor positive cells. The scale bar equals 25 pm. Data are from ref. 31.

3. Take extra care because DAB is a carcinogen. All residues should be inactivated with bleach prior to discarding. It is also very hygroscopic, so the bottle should be warmed at room temperature before opening to avoid moisture.

4. The brain or brain blocks should be placed on the filter paper in such a way that the cutting plane is parallel to the paper base. For coronal sections, we routinely separate the cerebrum and the cerebellum by cutting perpendicularly at the level of the colliculi, and stick each part to the paper by the cutting plane. Brain blocks can be similarly prepared by cutting through different planes as desired.

5. For coronal sections, a whole adult rat brain can be collected into 3 series of 5 tubes each. The first set of tubes, labeled A1-A5 contains alternating serial sections from the olfactory bulb to the fimbria of the hippocampus. The second set, labeled B1-B5 contains the sections from the rest of the cerebrum. The third set, tubes C1-C5 contains the cerebellar sections. Thus, if one tube of each set is used, a whole representation of the brain is present in a single experiment. Each tube contains enough sections to be hybridized with 3 probes. In this way, one adult rat brain can be hybridized with 15 different probes.

6. If the slides are to be used for emulsion photography, the sections should be mounted in the lower 2/3 of the slide to ensure a uniform emulsion thickness.

7. Check that there is absolutely no light sources other than the safe light in the room where the dipping is being perform. Take care to cover all possible sources of light from the water baths with black tape, and avoid static electricity sparks. Advertise very clearly with signs outside the room that there is an experiment running to avoid anybody to go in.

8. The ethanol solutions used in these steps should not contain ammonium acetate because otherwise the stain gets out of the sections.

9. The temperature and the time of incubation depends on the specific antibody used and also on antibody dilution. We generally incubate overnight at 4°C, but in many cases, this has to be empirically determined.

10. Dilution of secondary antibody, incubation time, and temperature are specified in the ABC staining kit instructions, but sometimes has to be optimized empirically.

11. It is important to stop the reaction when the brown color resulting from DAB oxidation is still light, so that the hybridization grains after emulsion photography can still be seen over the immune background color.

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