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Medical Research Coordination Center - PROTOCOL1

Protocol 1

Silver Acetate Autometallography (AMG)

 Responsible author: Univ.-Prof. Dr. Gerhard W. HACKER, Medical Research Coordination Center (MRCC) and Institute of Pathological Anatomy, Landeskliniken Salzburg (Salzburg Federal Hospital), Zentralbereich Medizin, and University of Salzburg, c/o Muellner Hauptstr. 48, A-5020 Salzburg, Austria, EU. Email-contact: g.hacker@lkasbg.gv.at


In the early eighties, a series of papers were published by Gorm DANSCHER, Aarhus, Denmark, to introduce a reliable and easy-to-handle technique for light microscopical and ultrastructural autometallographic (AMG) studies. Specific methods were developed to demonstrate endogenous zinc pools in synaptic and secretory vesicles; exogeneous mercury, silver bound as sulfide or selenide crystals, in lysosomes; and tissue-bound gold ions resulting from medicamented aurothiocompounds. It was demonstrated that gold ions chemically bound in the lysosomes had to be reduced to metallic gold before they could be silver-amplified. This understanding made it feasible to apply AMG amplification for magnifying colloidal gold particles in thick, semithin and ultrathin sections. As a result, the highly sensitive in situ colloidal gold-labeled detection of peptides, proteins, and amines by immunohistochemistry (immunogold-silver staining (IGSS), carbohydrates by lectin histochemistry, and DNA and RNA by in situ hybridization, in situ PCR and in situ 3SR techniques were born.

Silver Acetate AMG was developed and optimized during research visits of Gerhard W. HACKER at the University of Uppsala, Sweden, in joint collaboration with Gorm DANSCHER (Aarhus, DK) and Lars GRIMELIUS (Uppsala, S), from 1984 to 1988. Dr. Hacker would like to gratefully acknowledge the close friendship with these outstanding scientists. The technique published by us for the first time allowed silver enhancement of immunogold-silver staining under microscopical control and therefore can be used to yield optimal signal-to-noise ratio. Although the principle of silver acetate AMG is being used in a number of commercially available "light insensitive silver enhancement kits", it is more economical to perform it following our protocol.

Silver Acetate AMG can be applied for immunocytochemical gold-silver staining (immunogold-silver staining = IGSS; Nanogold-silver staining), as well as for lectin histochemistry, in situ hybridization, in situ PCR and the detection of metallic gold and silver, and sulfides and selenides of mercury, silver and zinc.

Original reference:
Hacker G.W., Grimelius L., Danscher G., Bernatzky G, Muss W., Adam H., Thurner J.: Silver acetate autometallography: an alternative enhancement technique for immunogold-silver staining (IGSS) and silver amplification of gold, silver, mercury and zinc in tissues. J. Histotechnol., 11: 213-221 (1988).

Review:
Hacker G.W., Danscher G.: Recent advances in immunogold-silver staining - autometallography. Cell Vision 1 (2), 102-109 (1994).

The following protocol outlines the application for gold label detection (colloidal gold, Nanogold):

  1. Solutions A and B should be freshly prepared for every run. Solution A: Dissolve 80 mg silver acetate (code 85140; Fluka, Buchs, Switzerland) in 40 mL of glass double-distilled water. (Silver acetate crystals can be dissolved by continuous stirring within about 15 min.)
  2. Citrate buffer: Dissolve 23.5 g of trisodium citrate dihydrate and 25.5 g citric acid monohydrate in 850 mL or deionized or distilled water. This buffer can be kept at 4C for at least 2-3 weeks. Before use, adjust to pH 3.8 with citric acid solution.
  3. Solution B: Dissolve 200 mg hydroquinone in 40 mL citrate buffer.
  4. Enhancement solution: Just before use, mix solution A with solution B.
  5. Silver amplification: Place the slides vertically in a glass container (preferably with about 80 mL volume and up to 19 slides; Schiefferdecker-type) and cover them with the mixture of solutions A and B. Staining intensity can be checked in the light microscope during the amplification process, which usually takes about 5-20 min, depending on primary antibody or nucleic acid probe concentration, incubation conditions, and the amount of accessible antigen or nucleic acid sequence in question.
  6. Photographic fixer (e.g., Agefix, Agfa Gevaert, Leverkusen, FRG; diluted 1:20) has been used in combination with colloidal gold to stop the enhancement process immediately. For this, slides are left in the fixer solution for about 1 min. Alternatively, a 2.5% aqueous solution of sodium thiosulfate can be used. It has, however, turned out that both these fixing treatments can be harmful when working with labeled tyramides or with Nanogold. We therefore now recommend to stop enhancement simply by washing in distilled water.
    URGENT NOTE: If you perform enhancement of gold label fur immunohistochemistry or in situ hybridization, and are using Nanogold instead of colloidal gold, it is crucial that slides are NOT dipped into sodium thiosulfate
    solution. Instead, the development process should be stopped by simply washing the sections in distilled water (several changes). (Sodium thiosulfate would remove the black  Nanogold-silver/gold staining already obtained.)
  7. After stopping the enhancement process, slides can be examined in a light microscope more carefully. If staining intensity is still too low, wash slides for one more time in double-distilled water and develop further in enhancement solution.
  8. After washing in distilled water, sections can be counterstained with nuclear fast red ("Kernechtrot"), and/or hematoxilin and/or eosin. For demonstrating nerve fibers or nerve cells, a light hematoxilin counterstain alone is a good choice. For good overall counterstaining, a combination of the three stains, in which the hematoxilin stain is carried out in a light version, appears adequate.
  9. After washing, dehydration in graded alcohols and clearing in xylene, DPX (BDH Chemicals, Poole, UK) or Permount are the preferred mounting media. Avoid using Eukitt! In our experience, the latter changes the intensity of silver stains after optimal development, sometimes even after weeks and months.


Some related REFERENCES    

last modified: 15.12.99 00:06:15
by:
Univ.-Prof.Dr. Gerhard W. Hacker G.Hacker@lkasbg.gv.at
and Dr. Mag. Heidi Bartel H.Bartel@lkasbg.gv.at

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