Materials
H-Thymidine, specific activity of 2.0
- curie/millimole
- Onion sets, jars and toothpicks
- Alcohol-acetic acid fixative
- Materials for feulgen reaction (Exercise 2.4)
- Paraffin embedding, sectioning equipment
- Kodak Nuclear Track Emulsion (or equivalent)
- Darkroom
- Water bath at 42° C
- Kodak D19 Developer
- Kodak Fixer
- Xylol, Permount and coverslips
- Microscope
Procedure
- Carefully read and follow all precautions for the safe handling of radioactive materials (Appendix H).
- Place toothpicks into the sides of an onion and set the onion into the top of a small jar or beaker. Fill the jar with water and allow the onion to begin root development (3- 7 days). Mild aeration of the water will assist in the growth of the roots.
- Set up another beaker, containing a 10 µ /ml solution of H-thymidine such that there are approximately 10 µc/ml, transfer the onion and its growing roots to the jar containing the radioactive thymidine.
- Allow the roots to stay in the radioactive solution for 1 hour at room temperature.
- Remove the onion and hold the bulb over a beaker containing water. Rinse the onion roots by dipping several times in the beaker. Transfer the bulb to yet another beaker of aerated water and allow to remain in this beaker for four hours.
- Cut off the roots tips and fix in alcohol-acetic acid (3:1) overnight. Change the fixative after the first four hours.
- Wash the roots in water for several minutes and place in 1N HCl at 60° C for 12 minutes.
- Stain the root tips with the Feulgen Reaction as given in Steps 3.b-3.e of Exercise 2.5.
- Dehydrate the root tips, embed in paraffin and section at 10-15 microns. Mount on microscope slides.
- Deparaffinize in xylol and rehydrate sections by passing first through a series of alcohols and finally in two changes of water.
- In a dark room 3 melt some liquid autoradiographic emulsion at a water bath at 42° C.
- Place two slides back-to-back and dip slowly into the melted emulsion. Remove, allow to drain and place in an appropriate light-proof container and allow them to dry in a vertical position.
- When dry, place the slides into an opaque slide box containing drierite. Wrap in aluminum foil and place in a refrigerator.
- The slides must stay in the refrigerator until a proper exposure has been made. This can vary from 5 days to over two weeks. After one week, a trial slide should be developed using Steps 15 and 16 below. Examine the slide at 10X with a bright field microscope and look for the presence of black silver grains located over the cells. A correct exposure is determined by the appearance of silver grains over the cells, but few or no silver grains located in areas without cells. If the appearance of the trial slide is correct, then the remainder should be processed immediately. Repeat an additional trial slide in 2-3 days, and repeat every three days thereafter until an adequate exposure is obtained.
- Develop the autoradiogram emulsion in the darkroom as follows:
- Develop in Kodak D-19 Developer at 20° C for 3 minutes.
- Wash for 10 seconds in distilled water.
- Fix with Kodak Fixer for 3 minutes.
- Wash in running water for 15 minutes.
- Dehydrate by placing in 95% alcohol for 3 minutes.
- Place in 100% alcohol for 3 minutes.
- Clear in two changes of xylol for 3 minutes each.
- Mount coverslip with Permount.
- Draw and label the autoradiograms in the space provided on the following page. Calculate the percent of cells actively undergoing DNA synthesis during the time of exposure to radioactive thymidine.
Note that the tissue and the silver grains are in different planes of focus and you will need to constantly switch focus from one plane to the other.
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Drawing of thymidine incorporation
Notes
This procedure involves the incorporation of a radioactive substance into a cell, and subsequent detection of that material through the use of a photographic emulsion. The primary source used for cell biology is an organic molecule containing tritium, the radioactive form of hydrogen. Radioactive carbon, phosphorous and iodine are occasionally utilized, but tritium has inherently more resolution than any of the others.
Tritiated thymidine (H-thymidine) is often used, for example, to study the synthesis and location of DNA. Thymidine is a soluble base which is specific to DNA. It is incorporated into the macromolecular structure of DNA during synthesis and replication of the chromosomes. Upon fixing the cells for standard histological examination, the DNA molecules (with their incorporated, radioactive, thymidine) are precipitated or cross-linked as permanent parts of the cell. Un-incorporated thymidine is removed from the cell, as it remains soluble and is disposed of in the tissue washing procedures.
When the tissues are sectioned and applied to a glass slide, they will contain radioactive nuclei, but only those nuclei that were in the S phase of division during the exposure of the cells to H-thymidine. Radioactive sources can not be detected directly, but if a photographic emulsion is applied directly over the section, it will become exposed by the radioactive source.
When the photographic emulsion is subsequently processed (i.e. developed), the exposed portions of the emulsion will contain reduced silver grains in direct proportion to the amount of radiation being given off beneath it (in the nuclei of our example).
If the exposed, developed slides are now examined with a microscope, there will be two layers of interest. Focusing the microscope on the tissue itself will give a view of the tissue and cell architecture. If the focus of the microscope is moved upward, however, the cells will go out of focus, exactly as the photographic emulsion comes into focus. Within the emulsion will be areas of reduced silver grains and clear areas containing no silver grains. By alternately focusing on the tissue and the emulsion, those nuclei that are radioactive can be readily identified.
If desired, the number of silver grains could be counted to give a quantitative measure of H- thymidine incorporation (and thus DNA synthesis), although this is a rather complex procedure to control, with any significant accuracy.
