Silver staining

Camillo Golgi perfected silver staining for the study of the nervous system. Although the exact chemical mechanism by which this occurs is unknown,{{cite journal |author= Golgi C|title= Sulla struttura della sostanza grigia del cervello |journal= Gazzetta Medica Italiana (Lombardia) |volume= 33|pages=244–246|year= 1873}} Golgi's method stains a limited number of cells at random in their entirety.{{cite journal |author= Grant G|title= How the 1906 Nobel Prize in Physiology or Medicine was shared between Golgi and Cajal |journal= Brain Res Rev |volume= 55|issue=2 |pages=490–498|date=Oct 2007 |pmid= 17306375 |doi= 10.1016/j.brainresrev.2006.11.004|s2cid= 24331507 }}

Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels.{{cite journal |vauthors=Kerenyi L, Gallyas F |title= Über Probleme der quantitiven Auswertung der mit physikalischer Entwicklung versilberten Agarelektrophoretogramme |journal= Clin. Chim. Acta |volume= 47|pages= 425–436|year=1973 |doi= 10.1016/0009-8981(73)90276-3|pmid= 4744834 |issue= 3}} The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports.{{cite journal |author= Switzer RC 3rd, Merril CR, Shifrin S|title= A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. |journal= Anal. Biochem. |volume=98|issue=1 |pages=231–237|date=Sep 1979 |pmid= 94518 |doi= 10.1016/0003-2697(79)90732-2}}

Classical Coomassie brilliant blue staining can usually detect a 50 ng protein band; silver staining increases the sensitivity typically 50 times.

Many variables can influence the color intensity and every protein has its own staining characteristics; clean glassware, pure reagents, and water of highest purity are the key points to successful staining.{{cite journal |vauthors=Hempelmann E, Schulze M, Götze O |title= Free SH-groups are important for the polychromatic staining of proteins with silver nitrate |journal= Neuhof V (Ed)Electrophoresis '84, Verlag Chemie Weinheim 1984|pages=328–330|year=1984}}

Some cells are argentaffin. These reduce silver solution to metallic silver after formalin fixation. Other cells are argyrophilic. These reduce silver solution to metallic silver after being exposed to the stain that contains a reductant, for example hydroquinone or formalin.

Silver nitrate forms insoluble silver phosphate with phosphate ions; this method is known as the Von Kossa Stain. When subjected to a reducing agent, usually hydroquinone, it forms black elementary silver. This is used for study of formation of calcium phosphate particles during bone growth.

Silver staining aids the visualization of targets of interest, namely intracellular and extracellular cellular components such as DNA and proteins, such as type III collagen and reticulin fibres by the deposition of metallic silver particles on the targets of interest.{{cite journal |vauthors=Schwint OA, Labraga M, Cervino CO, Haffar M, Sequeiros PH, Marcos HJ |display-authors=3|title=A modification of the staining technique of reticular fibres for image analysis of the cardiac collagen network |journal=Cardiovasc. Pathol. |volume=13 |issue=4 |pages=213–20 |year=2004 |pmid=15210137 |doi=10.1016/S1054-8807(03)00153-4 }}

Pseudomonas,{{cite journal |vauthors=Barnini S, Dodi C, Campa M |title=Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa |journal=Lett. Appl. Microbiol. |volume=39 |issue=3 |pages=274–7 |year=2004 |pmid=15287874 |doi=10.1111/j.1472-765X.2004.01576.x |doi-access=free }} Legionella, Leptospira, H. pylori, Bartonella and Treponema, and fungi such as Pneumocystis, Cryptococcus, and Candida are organisms that are stained with silver.{{citation needed|date=August 2012}}

Silver staining is used in karyotyping. Silver nitrate stains the nucleolar organization region (NOR)-associated protein, producing a dark region wherein the silver is deposited and denoting the activity of rRNA genes within the NOR. Human chromosomes 13, 14, 15, 21, and 22 have NORs, which increase the silver stain activity by at least 50 times.{{citation needed|date=August 2012}}

Silver staining is used to stain gels. The silver stain of proteins in Agarose gels was developed in 1973 by Kerenyi and Gallyas.{{cite journal | pmid = 4744834 | volume=47 | title=[Errors in quantitative estimations on agar electrophoresis using silver stain] | date=September 1973 | journal=Clin. Chim. Acta | pages=425–36 | vauthors=Kerényi L, Gallyas F | issue=3 | doi=10.1016/0009-8981(73)90276-3 }} Later it was adapted to polyacrylamide gels used in SDS-PAGE,{{cite journal | pmid = 92027 | volume=76 | title=Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain | pmc=411569 | year=1979 | journal=Proc Natl Acad Sci U S A | pages=4335–9 | vauthors=Merril CR, Switzer RC, Van Keuren ML | issue=9 | doi=10.1073/pnas.76.9.4335 | bibcode=1979PNAS...76.4335M | doi-access=free }}{{citation|surname1=R. C. Switzer, C. R. Merril, S. Shifrin|periodical=Anal Biochem|title=A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels|volume=98|issue=1|pages=231–237|pmid=94518|date=September 1979|language=de|doi=10.1016/0003-2697(79)90732-2

}}{{cite journal | vauthors = Rabilloud T | display-authors = etal | year = 1988| title = Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. | journal = Electrophoresis | volume = 9 | issue = 6| pages = 288–291 | pmid = 2466660 | doi = 10.1002/elps.1150090608 | s2cid = 33007991 }}{{cite journal | vauthors = Rabilloud T | year = 1992 | title = A comparison between low background silver diammine and silver nitrate protein stains. | journal = Electrophoresis | volume = 13 | issue = 7| pages = 429–439 | pmid = 1425556 | doi = 10.1002/elps.1150130190 | s2cid = 43084621 }}{{cite book | vauthors = Lelong C, Chevallet M, Luche S, Rabilloud T | chapter = Silver Staining of Proteins in 2DE Gels | year = 2009 | title = Two-Dimensional Electrophoresis Protocols | url = https://hal.archives-ouvertes.fr/hal-00377752/file/Silverstainchapter.pdf| series = Methods Mol Biol. | volume = 519 | pages = 339–350 | pmid = 19381593 | doi = 10.1007/978-1-59745-281-6_21 | isbn = 978-1-58829-937-6 | s2cid = 52820065 }} and also for staining DNA or RNA.{{cite journal | vauthors = Blum H, Beier H, Gross HJ | year = 1987| title = Improved silver staining of plant protein, RNA & DNA in PAA gels. | journal = Electrophoresis | volume = 8 | pages = 93–99 | doi = 10.1002/elps.1150080203 | s2cid = 84471792}} The glycosylations of glycoproteins and polysaccharides can be oxidised by a 1-hour pre-treatment with 0.1% periodic acid at 4 °C, which improves the binding of silver ions and the staining result.{{cite journal | vauthors = Dubray G, Bezard G | year = 1982| title = A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels | journal = Anal. Biochem. | volume = 119 | issue = 2| pages = 325–329 | pmid = 6176144 | doi = 10.1016/0003-2697(82)90593-0 }}

First, the proteins are denatured in the gel by a fixative solution of 10% acetic acid and 30% ethanol and precipitated, at the same time the detergent (mostly SDS) is extracted. The diffusion of the proteins is thus significantly reduced. After repeated washing with water, the gel is incubated in a silver nitrate solution. Silver ions bind to negatively charged side chains of the proteins. Excess silver ions are then washed off with water. In the final development step, the silver ions are reduced to elemental silver by addition of alkaline formaldehyde. This stains the sites where proteins are present, brown to black.

The intensity of the staining depends on the primary structure of the protein. Furthermore, the cleanliness of the vessels used and the purity of the reagents influence the silver stain.E. Hempelmann, M. Schulze, O. Götze: Free SH-groups are important for the polychromatic staining of proteins with silver nitrate. In: V. Neuhof (Editor): Electrophoresis. Verlag Chemie, Weinheim, 1984, pp. 328–330. Common artifacts in silver stained gels are bands of keratin in the ranges of 54–57 kDa and 65–68 kDa{{cite journal | pmid = 6197906 | volume=135 | title=Protein contaminants of sodium dodecyl sulfate-polyacrylamide gels | year=1983 | journal=Anal Biochem | pages=470–4 | vauthors=Ochs D | issue=2 | doi=10.1016/0003-2697(83)90714-5 }} as a contamination of the sample prior to the electrophoresis.

There are several silver stains incorporating methenamine, including:

• Grocott's methenamine silver stain, used widely as a screen for fungal organisms.
• Jones' stain, a methenamine silver–periodic acid–Schiff that stains for basement membrane, availing to view the "spiked" GBM associated with membranous glomerulonephritis.

Image:Histoplasma in granuloma gms.jpg|A silver stain (GMS) demonstrating the fungus Histoplasma (black round balls) in a liver biopsy.

File:PAGE_AgStain_Microsat.jpg|DNA samples amplified using PCR. Samples have been visualized using silver staining.

File:RBC Membrane Proteins SDS-PAGE gel.jpg|RBC membrane proteins separated by SDS-PAGE and silver-stained.{{cite journal |vauthors=Hempelmann E, Götze O | title =Characterization of membrane proteins by polychromatic silver staining| journal = Hoppe-Seyler's Z Physiol Chem| volume = 365| pages = 241–242| year = 1984 }}

{{reflist}}

• {{LoyolaMedEd|Histo/practical/stains/hp2-55.html}}
• [https://web.archive.org/web/20100714145637/http://www.biomalpar.org/updatedMethods_In_Malaria_Research_5thedition.pdf] Hempelmann E. SDS-Protein PAGE and protein detection by silverstaining and immunoblotting of Plasmodium falciparum proteins. in: Moll K, Ljungström J, Perlmann H, Scherf A, Wahlgren M (eds) Methods in Malaria Research, 5th edition, 2008, 263-266

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Category:Electron microscopy stains

Category:Genetics techniques

Category:Staining

Category:Stained glass