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Designed to reduce the fading of the fluorescence of dyes used for labelling biological specimens.
In fluorescence microscopy, the fluorescence is stimulated by high intensity UV and/or visible light. Absorption of the light creates an excited state of the dye (usually a singlet state) and this leads to fluorescence. However, the excited state of the dye may undergo chemical reactions which lead to its destruction as evidenced by the fading or bleaching of the fluorescence and consequent loss of the image, or in the case of assays, a change in signal intensity during measurement.
There is no universal anti-fadent; due to the varied nature of biological material the fluorescence fading of fluorochromic conjugates is a complex process.
The general anti-fadents are AF1, AF2 and AF3. AF87 is a specialist immersion oil-based anti-fadent.
Guide to Non-Hardening Mountants
- AF1 A glycerol-PBS solution contains an amine antifadent.
- AF1 plus DAPI contains both an amine antifadent and the DNA stain, DAPI.
- AF2 A glycerol solution contains an amine antifadent that allows you to choose your own buffer solution.
- AF4 A glycerol solution of n-propyl gallate.
- AF3 is a PBS solution containing an amine-based antifadent.
- CFPVOH is an aqueous solution of poly (vinyl alcohol). Provided care is taken not to allow the water to evaporate, it will remain fluid. Retardation of photobleaching is effected by the addition of the aqueous antifadent solution AF100.
- AF100 Is a PBS solution of an amine-based antifadent that if used as an additive to CFPVOH (1 part AF100 to 9 parts CFPVOH). Provided care is taken not to allow the water to evaporate, it will remain fluid.
- AFR3 is a PBS solution containing a NEW non-amine, non phenolic antifadent.
Guide to Hardening Mountants
Hardening mountants are aqueous solutions containing a polymer such a poly (vinyl alcohol) (PVOH). When a few microlitres of these solutions are pipetted onto a microscope slide and a coverslip applied, evaporation of water slowly takes place and the formation of a stable film results which immobilises the coverslip.
A variety of hardening mountants is available which provide films possessing a range of hardness. They are based on water-soluble polymers such as poly(vinyl alcohol(PVOH) and poly (vinyl pyrrolidone)(PVP).
Why are aqueous glycerol solutions of PVOH containing antifadents inherently unstable?
PVOH is produced by hydrolysis of poly(vinyl acetate) and most commercial samples of PVOH contain residual (unhydrolysed) acetate groups. These groups undergo hydrolysis, often accelerated by the added antifadents, during storage and this can cause a change in pH and more usually gellation.
This latter process leads to an unpredictable shelf-life. By making up small volumes of PVOH solution containing the required amount of antifadent solution, you have materials of consistent composition and performance as well as making better use of your purchased materials.
- AF200 is a glycerol solution containing an amine-based antifadent.
- AF300 is a glycerol solution containing a phenolic type of antifadent.
Film Forming Polymer Solutions
- Tris-MWL 4-88 is a classical, popular mountant solution based on Mowiol? 4-88, glycerol, water and tris-amine buffer. Following evaporation of the water a film of weak to medium strength is formed. To have effective reduction in photobleaching, it should be used with either AF100, AF200 or AF300.
- CFPVOH is an aqueous solution of poly (vinyl alcohol) and is designed to be used with the glycerol-based antifadent solutions AF200 or AF300. Following evaporation of the water films of medium hardness are produced.
- PVP plus antifadent is an aqueous solution of poly (vinyl pyrrolidone) containing an amine-based antifadent. The solutions are stable over long periods e.g. in excess of five years.
- CFPVOH plus antifadent is an aqueous solution of a carefully selected PVOH containing an amine-based antifadent and has a shelf life 6 months.
Film Forming Polymer Solutions
- CFPVOH is an aqueous solution of poly (vinyl alcohol). Retardation of photobleaching is effected by the addition of the aqueous antifadent solution AF100. (1 part AF100 to 9 parts CFPVOH)