Anti-fadent Solutions reduce the photo-bleaching, 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. For work at high magnification, a non-fluorescent immersion oil is available
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 single 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.
To overcome these problems, use anti-fadent (anti-bleaching) solutions as mounting media when specimens are examined by fluorescent microscopy or other detection systems and as additives for assays.
The most frequently used solution is AF1 which is an anti-fadent (anti-bleaching agent) contained in a glycerol PBS (phosphate-buffered saline) solution and is particularly useful for examining tissue sections.
AF2 solution contains the anti-fadent in glycerol which enables users to choose their own buffer, and AF3 is the anti-fadent in PBS solution. AF3 is particularly useful for examining live cells.
AF87 is a non fluorescent immersion oil containing anti-fadent. CFPVOH is an aqueous solution of poly (vinyl alcohol) for use as a solid mountant. AF100 is a solution of anti-fadents (anti bleaching agents) for use with CFPVOH when fading (bleaching) of specimens in a solid mountant is a problem.
None of the anti-fadents is based on p-phenylenediamine. All solutions may be stored at room temperature.
Choosing the Best Anti-fadent
These are the critical decision factors which will help you select the most appropriate mountant:
- Do you need a Hardening or a Non Hardening medium?
- If your system is Glycerol tolerant than choose a Glycerol Based product, if not then use a Glycerol Free solution
- 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
** Store at room temperature
A more detailed account of our anti-fadent products can be found in the Technical Data Sheets below. The following is a brief overview which we hope helps you to decide on the best solution for your application.
If you are still unsure about which mountant to use, please don't hesitate to contact us, either by phone or email. We have technical staff on hand who are happy to answer the simplest, or the most challenging of questions.
Guide to Non-Hardening Mountants
- AF1 A glycerol-PBS solution contains an amine anti-fadent SDS - AF1
- AF1 plus DAPI contains both an amine anti-fadent and the DNA stain, DAPI
- AF2 A glycerol solution contains an amine anti-fadent that allows you to choose your own buffer solution SDS - AF2
- 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 muylti-photon confocal niche since it acts as an efficient cleaning 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 it can be seen that 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 SDS - CFM-1
- CFM-1 plus AF is a glycerol-based solution having a refractive index of ~1.52 (at room temperature) that contains an amine anti-fadent SDS - CFM-1 + AF
- 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 SDS - CFM-2
- CFM-3 This glycerol-based anti-fadent contains a phoenlic anti-fadent of neutral pH and a refractive index of ~1.52 which also acts as a Clearing Solution enabling visualisation of fluorochromes deep within the sample SDS - CFM-3
- CFMR2 This product was designed for use with samples labelled with GFP. It contains a unique anti-fadent which does not de-oxygenate the solution which is an essential property if the GFP is not to bleach SDS - CFMR2
- AF3 is a PBS solution containing an amine-based anti-fadent SDS - AF3
- 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 anti-fadent solution AF100
- AF100 Is a PBS solution of an amine-based anti-fadent 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 SDS - AF100
- AFR3is a PBS solution containing a NEW non-amine, non phenolic anti-fadent SDS - AFR3
High Refractive Index Glycerol Free Solutions
AF87 is an immersion oil having a refractive index of 1.52 and contains an anti-fadent. It may be used as an immersion oil and also as a mountant. Since AF87 is an oil that is immersible with water, it is essential that specimens are dehydrated before application of the mountant. SDS - AF87
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 anti-fadents 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 anti-fadents, 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 anti-fadent 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 anti-fadent SDS - AF200
- AF300 is a glycerol solution containing a phenolic type of anti-fadent SDS - AF300
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 photo bleaching, it should be used with either AF100, AF200 or AF300 SDS - MWL4-88
- CFPVOH is an aqueous solution of poly (vinyl alcohol) and is designed to be used with the glycerol-based anti-fadent solutions AF200 or AF300. Following evaporation of the water films of medium hardness are produced SDS - CFPVOH
- PVP plus anti-fadent is an aqueous solution of poly (vinyl pyrrolidone) containing an amine-based anti-fadent. The solutions are stable over long periods e.g. in excess of five years SDS - PVP-1
- CFPVOH plus anti-fadent is an aqueous solution of a carefully selected PVOH containing an amine-based anti-fadent and has a shelf life 6 months
Film Forming Polymer Solutions
- CFPVOH is an aqueous solution of poly (vinyl alcohol). Retardation of photo bleaching is effected by the addition of the aqueous anti-fadent solution AF100. (1 part AF100 to 9 parts CFPVOH)
|Code||Title||Type||Pack Size||Availability||Price||Updated: 18-01-2022|
|IAF1||Citifluor anti-fadent||AF1||25mL||1 in stock||AU $67.00|
|EMS17970-100||Citifluor anti-fadent||AF1||100mL||2 weeks||AU $174.00|
|IAF2||Citifluor anti-fadent||AF2||25mL||2 weeks||AU $67.00|
|EMS17971-100||Citifluor anti-fadent||AF2||100mL||2 weeks||AU $174.00|
|IAF3||Citifluor anti-fadent||AF3||25mL||2 weeks||AU $70.00|
|EMS17972-100||Citifluor anti-fadent||AF3||100mL||2 weeks||AU $174.00|
|IAF87||Citifluor anti-fadent||AF87||10mL||2 weeks||AU $62.00|
|EMS17976-25||Citifluor anti-fadent||AF87||25mL||2 weeks||AU $99.00|
|EMSAF100-5||Citifluor anti-fadent||AF100||5mL||2 weeks||AU $24.00|
|EMS17980-10||Citifluor anti-fadent||CFM-1||10mL||2 weeks||AU $58.00|
|EMS17980-25||Citifluor anti-fadent||CFM-1||25mL||2 weeks||AU $92.00|
|EMS17981-10||Citifluor anti-fadent||CFM-1+Antifadent||10mL||2 weeks||AU $63.00|
|EMS17981-25||Citifluor anti-fadent||CFM-1+Antifadent||25 mL||2 weeks||AU $102.00|
|EMS17982-10||Citifluor anti-fadent||CFM-2||10mL||2 weeks||AU $58.00|
|EMS17982-25||Citifluor anti-fadent||CFM-2||25 mL||2 weeks||AU $92.00|
|EMS17970-125||Citifluor anti-fadent||AF1 + DAPI||15mL||2 weeks||AU $75.00|
|EMS17973-10||Citifluor anti-fadent||AF3||10mL||2 weeks||AU $106.00|
|EMS17973-25||Citifluor anti-fadent||AF4||25mL||2 weeks||AU $70.00|
|EMS17977-10||Citifluor anti-fadent||AF200||10mL||2 weeks||AU $70.00|
|EMS17977-25||Citifluor anti-fadent||AF300||10mL||2 weeks||AU $70.00|
|EMS17977-50||Citifluor anti-fadent||PVP-1||25mL||2 weeks||AU $70.00|
|EMS17977-100||Citifluor anti-fadent||PVP-1 + Antifadent||25mL||2 weeks||AU $80.00|
|EMS17977-150||Citifluor anti-fadent||MWL4-88||25mL||2 weeks||AU $70.00|
|EMS17979-10||Citifluor anti-fadent||CFMR2||10mL||2 weeks||AU $88.00|
|ICFM3-10||Citifluor anti-fadent||CFM3||10mL||2 weeks||AU $62.00|
|ICFM3-25||Citifluor anti-fadent||CFM3||25mL||2 weeks||AU $101.00|
|EMS17979-40||Citifluor anti-fadent||PF||25mL||2 weeks||AU $134.00|
|EMS17979-50||Citifluor anti-fadent||PF/HRF||10mL||2 weeks||AU $96.00|