Citifluor™ Antifadent Solutions

Mountant Media Containing Antifadents – The Antidote For Photobleaching

What is an Antifadent Solution?

Antifadent Solutions reduce the photobleaching or fading of the fluorescence of dyes used for labelling biological species. The fading of fluorochrome dyes is a particular problem in fluorescence microscopy, such as in immunofluorescence studies. Fluorescent dyes are also used as cell markers, for following the uptake and release of calcium from cells for example, and for characterising cell surfaces. Non-fluorescent immersion oil is available for work at high magnifications.

In fluorescence microscopy, the fluorescence is stimulated by high intensity UV or visible light. Absorption of the light populates 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 leads 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.

Using antifadent (anti-bleaching) solutions as mounting media are ideal to overcome the problems of examining samples by fluorescent microscopy or other detection systems and as an additives for assays.

The most frequently used solution is AF1 which is an antifadent (anti-bleaching agent) contained in a glycerol PBS (phosphate-buffered saline) solution and is particularly useful for examining tissue sections.

AF2 solution contains the antifadent in glycerol which enables users to choose their own buffer, and AF3 is the antifadent in PBS solution. AF3 is particularly useful for examining live cells.

AF87 is a non-fluorescent immersion oil containing antifadent. CFPVOH is an aqueous solution of poly (vinyl alcohol) for use as a solid mountant. AF100 is a solution of antifadents (anti bleaching agents) for use with CFPVOH when fading (bleaching) of specimens in a solid mountant is a problem.

These antifadents are not based on p-phenylenediamine. All solutions may be stored at room temperature.

Choosing the Best Antifadent

These are the critical decision factors which will help you select the most appropriate mountant:

1. Do you need a Hardening or a Non Hardening medium?
2. If your system is Glycerol tolerant than choose a Glycerol Based product, if not then use a Glycerol Free solution
3. These high refractive index mountant solutions are to be used where you wish to obviate the effects of spherical aberration (caused by a mismatch between the refractive index of the glass of the coverslip and the mountant medium) which leads to a loss of resolution of your images.

Table 1. Types of antifadent available

OVERVIEW OF APPLICATIONS

Non-Hardening Mountants: Use of Non-Hardening Citifluor antifadents

Glycerol Based

• 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

High Refractive Index Glycerol Based Solutions

These solutions have a refractive index that matches that of the glass of the coverslip thereby minimising the effects of spherical aberration. Such mountants are invaluable for imaging the internal structures of samples by techniques such as single multi-photon confocal niche since it acts as an efficient Clearing Solution (clearing occurs within a few minutes – Sean Speese, Oregon Health and State University, Personal Communication). The beneficial effects of these reagents can be seen in the two figures below where high resolution images have been obtained at a significant depth within the mountant solution. Additional features of these solutions includes their total water miscibility, they are non-odorous and do not quench the fluorescence of fluorochromes (unlike thiodiethanol, another proposed high refractive index mountant) and the solutions are stable for long periods (at least 1 year).

• CFM-1 is a glycerol-PBS buffered solution having a refractive index of ~1.52 which can be used for transmission microscopy as well as with epifluorescence microscopy
• CFM-1 plus AF is a glycerol-based solution having a refractive index of ~1.52 (at room temperature) that contains an amine antifadent
• CFM-2 is a glycerol-tris amine buffered solution having a refractive index of ~1.52 (at room temperature) and a pH of ~8.5
• CFM-3 – This glycerol-based antifadent contains a phoenlic antifadent of neutral pH and a refractive index of ~1.52 which also acts as a Clearing Solution enabling visualization of fluorochromes deep within the sample
• CFMR2 –This product was designed for use with samples labeled with GFP. It contains a unique antifadent which does not de-oxygenate the solution which is an essential property if the GFP is not to bleach

Glycerol Free

• 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

High Refractive Index Glycerol Free Solutions

AF87 is an immersion oil having a refractive index of 1.52 and contains an antifadent. It may be used as an immersion oil and also as a mountant. Since AF87 is an oil that is immiscible with water, it is essential that specimens are dehydrated before application of the mountant.

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.

Glycerol Based

• 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

Glycerol Free

• 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)