Coating the Grids With Normal Serum see Note

1. Use an equivalent dilution of normal serum as that being used in the experiment or test as described in Subheadings 3.1.-3.3.

2. Follow the rest of the procedures as described in Subheadings 3.1.-3.3.

4. Notes

1. Originally called serologically specific electron microscopy (11), but variously known as antiserum coated grids, serum activated grids or "trapping."

2. Personal experience has shown that, although monoclonal antibodies can be used in ISEM, better results are more often obtained by using crude antiserum. Others have had the same experience (4). In addition antisera preserved in glycerol can be used successfully for detecting viruses by IEM (8).

3. Good results are obtained using a 1:500 dilution of antiserum but can also be achieved with higher dilutions (e.g., 1:1000) (1,2,4,6). Similarly although a 1:100 dilution of antiserum is used for "decorating" virus particles in the methods described here, higher or lower dilutions can be used to give the required amount of antibody 'decoration' to enable the user to be convinced that the technique has been successful.

4. Phosphate buffer pH range 6-7, 0.05-0.2 M has been found to be generally compatible for extracting most viruses. However, others, including Tris-HCl buffer (0.05-0.1 M, pH range 6.5-7.2), should be tried if results are unsatisfactory (4,11).

5. A wide range of drop sizes are cited in the literature between 5 and 50 ^L (4), but 20-^L drop is a good general size to use for convenience and reproducibility.

6. There are many different support films available but carbon is the most consistently reliable. Grids can be prepared in the laboratory or purchased commercially, already carbon-coated, from specialist suppliers.

7. Good results are obtained by using a 15-min incubation time at room temperature as a general rule. However, for some antiserum/virus combinations a lower temperature, for example, 4°C, and a longer incubation time, for example, 4 h to overnight, may be preferable for viruses that are phloem restricted and/or in low concentration within the plant. This also can increase sensitivity (11). If unsatisfactory results are experienced then optimum conditions will need to be determined by experiment.

8. Grids can be dried and stored after the antiserum coating step and can be used up to 6 wk later for trapping virus particles (2).

9. As a general rule leaf material is chosen but roots, petals, pollen, seeds (12), fruit, or even virus vectors (13,14) can be suitable. Choice will depend on the concentration of virus particles, which will differ within a plant and can depend on the virus being examined.

10. There are various methods available for extracting sap. For small amounts crush in buffer on a glass slide with a flattened glass rod (6), for larger samples grind with a pestle and mortar (8,11). If an abrasive is used, for example, Carborundum or Celite, the extract may need to be centrifuged to ensure that there is a clear supernatant. In this case the recommendation would be no longer than 1 min at 10,000 rpm (4).

11. Rinsing must be conducted accurately and consistently for good results to be obtained. The general principal is that the more you rinse the better the results (2).

12. There are always preferences as to which stain to use for any given virus. An aqueous solution of uranyl acetate (UA) at 2% is a good general stain but others should be tried if this does not give the results required (2). UA is incompatible with some buffers, especially phosphate, which is the buffer of preference in this chapter. To avoid precipitation, treated grids must be washed with distilled water before UA is used (1,4). In addition UA is toxic, so users should refer to the Hazard Data Sheet produced by the manufacturer before use.

13. Grids already negatively stained in UA can be rinsed with distilled water, then phosphate buffer and then incubated with antiserum to achieve decoration, followed by re-staining with UA (2). However, personal experience has shown that this is more often than not unsuccessful and it is better to prepare a fresh grid rather than spend time on an unreliable method.

14. Treated grids can be stored in commercially available grid boxes, after staining, for later examination.

15. It has been observed that more virus particles can be found after coating with antiserum than before it (1), and it is thought that this is because coating the virus particles with the antibody binds them even more strongly to the grid, decreasing the loss of particles during the negative staining stage of the procedure (4).

16. The various antisera used to coat the grids can be used systematically (and separately) in the decoration process following this method.

17. To prepare a conventional EM grid, squash a piece of leaf material in 2% potassium phosphotungstate brought to pH 6.8 with 0.1 M potassium hydroxide on a glass slide using a glass rod with a flattened end. Place one drop of the sap extract on to a carbon-coated grid, dry with filter paper and examine in the TEM.

18. With high concentrations of virus, particles will form clumps before much coating with antibody has occurred. With lower concentrations of particles they become evenly coated before aggregating (4). For the latter, use the following method: dilute the sap with phosphate buffer, pH 6.5, until less than one rod shaped or filamentous particle or less than five spherical virus particles are seen per field of view at a magnification of 20,000 in a conventional preparation in the electron microscope (see Note 17). Then, follow the method as described.

19. Protein A plus antiserum coated grids can be stored for 12 mo or more at -20° C and still retain their activity (9).

20. These preparations can be bought commercially, either direct from the catalogue or using a customized conjugation service for labeling antibodies with gold particles, in a wide range of sizes suitable for different magnifications.

21. This process will represent the numbers of virus particles, which would be found by conventional electron microscopy (see Note 17) and therefore will contain far fewer compared to the trapped grid.

22. Serum from an animal that has not been used for specific antibody production (4). Alternatively, an unrelated antiserum can be used or buffer alone (4).

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