Silver Intensification for Immunocytochemistry
Silver intensification solutions
- Stock A = 114 mg citric acid + 342 mg sodium citrate in 6 ml deionized water (make fresh – do not store)
- Stock B = 0.5 g hydroquinone in 15 ml deionized water (make fresh – do not store)
- Stock C = 1% aqueous silver nitrate (may be stored indefinitely at room temperature wrapped in foil)
- Working solution = 5 ml A + 1 ml B + 1 ml C, in that order, made up immediately before use
- Stop solution = 5% acetic acid
- Optional: Slide warmer set at 30°C (Avoid cold surfaces. Temperatures below 24°C slow the reaction).
- Prepare silver intensification solutions. Stock solution C may be prepared in advance. Prepare stock solutions A and B separately prior to intensification and use within 1 hour.
- Use the working solution immediately as it lasts only 10 min. Add 25-50 microliters of solution per well in quick succession; a small disposable dropper works best. (Alternative: make 15 ml of working solution and process in a plastic slide cassette). Expose sections to the solution for 4-8 minutes in a dark location, such as under an aluminum cover. You needn’t work in absolute darkness, but the background decreases slightly if the intensification occurs away from bright light. The reaction is temperature sensitive and slows substantially below 24°C.
- Stop the reaction with a brief dip in 5% acetic acid.
- Wash for 10 minutes in deionized water and dry with an air canister.
- Cover slip in a medium of your choice.
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