Caution: Purity of water and cleanliness of glassware are crucial for optimal silver-staining results. Also, contact between metallic objects (for example, metal forceps) and the silver solution should be avoided.
Prepare the following reagents fresh daily except for the citrate buffer.
Citrate buffer: Dissolve 23.5 g trisodium citrate dihydrate and 25.5 g citric acid monohydrate in 850 ml distilled water. Check for microbial contamination before each use if not making fresh each time.
Solution A: Dissolve 100 mg silver acetate (Fluka Chemical Co., catalog # 85140) in 50 ml distilled water and keep under cover. To speed dissolving the fluffy powder, stir vigorously. The reagent may be used for an entire day if protected from light.
Solution B: Dissolve 250 mg hydroquinone in 50 ml citrate buffer that has been freshly adjusted to pH 3.8 using citric acid.
Caution: Fixation of the tissue in 1-2% glutaraldehyde is crucial since the low pH (3.8) developer used in this protocol may dissociate the previously labeled reagents from the tissue.
Place the slide for 5 min in solution B diluted to one-half strength with distilled water.
Transfer the slide to the developer (equal volumes of solution A and solution B, mixed just before use). Develop under cover at room temperature for 4 - 18 min. The degree of staining may be monitored periodically under a bright-field microscope, adjusted to low light intensity. If a more intense staining is desired, place the slide back in the developer. For certain studies, addition of crude gum arabic to a final concentration of 15% to the developer should slow the rate of silver accumulation and thereby make it more controllable. For prolonged incubation, keep under a dark box.
After a quick rinse in distilled water, place the slide in a commercial photographic fixative for 2 min.
Rinse with tap water for at least 5 min.
Counterstain with hematoxylin and eosin (paraffin and cryostat sections) or methylene blue (semi-thin resin sections), dehydrate, clear, and mount.
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