1. Fix the specimen in neutral buffered formalin and embed in wax. Cut into 3- to 5-^m sections.
2. Dewax the sections in xylene (two changes, 5 min each) and rehydrate through three changes of IMS to tap water.
3. Lugol's iodine/Hypo sequence (in the author's experience, this is only required when using Danscher's reagent, and not if a commercial silver enhancement kit is used). Immerse in Lugol's iodine (5 min) and remove the iodine by rinsing thoroughly in 5% sodium thiosul-fate, followed by washing in PBS.
4. Wipe excess liquid from the area around the sections and apply sufficient 5% heat-inactivated normal serum from the second antibody species to cover each entire section (15 min). If protein A-gold probes are used, flood the sections with BSA-PBS rather than normal serum. It is important to keep the sections covered and in a humid environment throughout all incubations, preferably using a purpose-made humidity chamber. Depending on the size of the section, approx 50-200 ^L of reagent is sufficient to cover each preparation.
5. Tip the slides, wipe away the normal serum from around the section and replace, without rinsing, with specific primary antibody for 1 h. The appropriate dilution of the primary antibody should be decided previously either by following the manufacturer's recommendations, or by titration assay—conducting a series of dilutions and determining which gives the best signal to noise ratio.
6. Rinse the slides in BSA-PBS, three changes of 2 min each.
7. Cover the sections with gold probe, again suitably diluted in BSA-PBS following either the manufacturer's recommendations or by titration assay.
8. Rinse the slides in BSA-PBS, three changes of 2 min each.
9. If weak antibody-antigen binding is suspected, the binding may be stabilized by fixation in 1% glutaraldehyde in PBS for 10 min.
10. Rinse sections in high-quality distilled water to remove all traces of chloride ions and other impurities that might contaminate the silver enhancing solution. Some commercial enhancement solutions are reported to be resistant to contamination; the user should evaluate this carefully, especially if high levels of nonspecific background are encountered.
11. If a commercial silver enhancement kit is used, make up enhancer immediately before use according to the manufacturer's instructions, especially with respect to times, temperatures and lighting conditions. Every 2 min, examine the progress of the enhancement using a bench microscope but be aware that some of the more sensitive enhancers, in particular Danscher's Reagent, may undergo spontaneous reduction when exposed to strong light. It may be useful to place a control slide continuously on the microscope stage; the lamp may then be turned on when required to examine the progress of enhancement (see Notes 2 and 3).
12. When the reaction has developed sufficiently, according to the investigator's preference, wash the slides first in distilled water and then in running tap water.
13. To prevent the silver intensification from fading, fix the reaction product with 5% sodium thiosulfate for 5 min and wash with tap water.
14. Counterstain as required; either 1 min in Gill's hematoxylin alone, 1% eosin alone, or hematoxylin followed by eosin will give good contrast with the black silver deposition.
15. Wash well in tap water, dehydrate in IMS, clear in xylene, and mount in a synthetic mounting medium.
16. Sites of antibody-antigen localization will appear black when viewed with transmitted white light (Fig. 2).
17. There are various ways of adjusting, improving and measuring the IGSS reaction product (see Notes 4-11).
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