Materials

1. An antibody of known specificity and species. It does not matter whether the antibody is monoclonal or polyclonal.

2. A gold probe, preferably 5 nm in diameter or less, coated with an immunological protein specific to the primary antibody. In principle, small gold particles produce a higher labeling intensity of the target antigen because of reduced steric hindrance. If the primary antibody is from a rabbit, gold coated with protein A or anti-rabbit antiserum may be used. If the primary antibody is from mouse, gold coated with protein G or an antibody raised against the correct isotype of immunoglobin G is used. If the antibody is biotinylated, gold complexed to either streptavidin or a monoclonal antibody to biotin is used. These will be referred to as the gold probe. In the context of the final result, it does not matter which probe is used as long as it reacts with the primary antibody.

3. Lugol's iodine.

4. 5% Sodium thiosulfate.

5. Phosphate-buffered saline (PBS: 0.01 M sodium phosphate, 0.5 M NaCl, pH 7.2, as suggested by Slot and Geuze (15).

6. Heat-inactivated serum from the second antibody species (not for use with the protein A-gold technique).

7. 1% Bovine serum albumin in PBS (BSA-PBS).

8. Commercial silver enhancement kit.

9. Double-distilled water.

10. Gill's hematoxylin.

11. Industrial methylated spirit (IMS).

12. Xylene.

13. Gum acacia (500 g/L in distilled water).

14. Trisodium citrate dihydrate.

15. Citric acid.

16. Hydroquinone (0.85 g/15 mL distilled water, freshly prepared).

17. Silver lactate (0.11 g/15 mL distilled water, freshly prepared).

18. Tris-buffered saline (TBS): 0.05 M Tris-HCl in isotonic (0.9%) saline, pH 7.6.

20. Calcium chloride.

21. Neutral buffered formalin.

22. 1% Glutaraldehyde.

23. 1% Eosin in distilled water.

24 Silver enhancement solution: the silver solution for enhancing the gold probes is prepared by mixing the following reagents in the order given and using immediately. All solutions are made in distilled water. Keep the silver lactate and the final mixture containing the silver lactate dark by wrapping the containers within foil and use in a darkroom. At the acid pH, silver lactate provides the source of silver ions necessary for deposition on the gold particles. The reducing agent for the reaction is hydroquinone and the gum acacia is included to prevent the silver from being reduced too rapidly ("autoreduction") which would lead to high background levels ("autonucleation").

25. Gum acacia (7.5 mL or 50 g/L) prepared by stirring overnight and filtering through gauze. Stock solutions may be kept in frozen aliquots.

26. Citrate buffer (10 mL) at pH 3.0. This consists of 23.5 g of trisodium citrate dihydrate and 2.5 g of citric acid monohydrate dissolved in 100 mL of distilled water.

27. Freshly prepared hydroquinone (15 mL).

28. Freshly prepared silver lactate (15 mL).

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