EM Autoradiography

1. Dissect salivary glands in saline solution as described in Subheading 3.1.1., step 2 and collect them in a separate cup in saline solution.

2. Transfer 10-15 glands into saline solution containing 3H-uridine and incubate for 5 min.

3. Fix the glands either in cold ethanol : acetic acid (3 : 1, v/v) for 15 min or in 45% acetic acid for 3-5 min.

4. Squash, stain, and dehydrate the glands as described in Subheading 3.1.1., steps 4-10.

5. Embed the glands in epoxy resin as described in Subheading 3.1.2., steps 1-7.

6. Check for 3H-uridine incorporation by coating the Araldite blocks with liquid photographic emulsion. Expose in the dark for 72-120 h and then develop them with Kodak D-19 developer (3 min).

7. Choose specimens with high levels of labeling and remove the emulsion with warm water.

8. Proceed from Subheading 3.1.2., step 8 continuing through to Subheading 3.1.4., step 4.

9. Assemble the grids with sections on a narrow strip of plexiglass using scotch tape. Prepare two to three similar strips with "empty" grids (i.e., without sections). These will be used to test the thickness and distribution of the emulsion.

10. Melt a portion of Ilford L-4 emulsion at 40°C in a dark room, dilute it with double-distilled water, and gently mix to homogeneity using a clean glass rod.

11. Cover "empty" grids with emulsion and check the distribution of silver grains under the EM. This test is to ensure that the emulsion gives a monolayer of halide silver crystals. If necessary, continue diluting the emulsion until the desired consistency is obtained. When working with photographic emulsion, follow the manufacturer's instructions.

12. Cover the grids having the sections with the same emulsion: Dip the plexiglass strip into the liquid emulsion, pull it out, and allow the emulsion to dry.

13. Expose the grids in a dark box for 2-2.5 mo.

14. Develop the preparations with Kodak D-19 developer (3 min).

15. Examine chromosome labeling under EM at 80 kV.

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