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Tips & Tricks
| Useful tips AF1 |
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In some cases, the reduction in the rate of PHOTOBLEACHING may be accompanied by a reduction in the initial intensity of the fluorescence signal. By diluting the AF1 solution with glycerol the reduction in the intensity of the fluorescence signal can oftentimes be mitigated. Dilution of AF1 with glycerol will increase its refractive index whereas dilution with water will reduce its refractive index. PVOH is produced by hydrolysis of poly(vinyl acetate) and most commercial samples of PVOH contain residual (unhydrolyzed) 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 gelation. 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. |
| Useful tip AF2, AF3 |
| If the viscosity of the AF2 solution is too high for your purpose, it may be reduced by the addition of either AF1 or AF3 solutions. |
Storage Tips for various Mounting Media
| Storage and Shelf-Life AF1 |
| AF1 solutions may be stored at room temperature and ideally between 5° and 15°C and out of strong sunlight. The cap of the bottle or if using the pipette supplied with the material, the cap which covers the pipette delivery point, should always be replaced after use as a matter of good practice. Samples stored under these conditions for 6 months have shown no apparent deterioration. If the AF1 solution is being used in an assay, a control experiment should always be carried out. |
| Storage and shelf-life AF2, AF3 |
| The solutions are of medium viscosity and are water-white in appearance. They may be stored at room temperature and ideally between 5° and 15°C and out of strong sunlight. The cap of the bottle or if using the pipette supplied with the material, the cap which covers the pipette delivery point, should always be replaced after use as a matter of good practice and also to prevent the ingress of water (due to the glycerol being hygroscopic). Samples stored under these conditions for 6 months have shown no apparent deterioration. If the AF2 solution is being used in an assay, a control experiment should always be carried out. |
| Storage and shelf-life AF4 |
| The mountant solution should be stored in brown glass bottles at temperatures between 5° and 15°C and shielded from strong sunlight. The cap of the bottle should always be replaced after use as a matter of good practice and to prevent the absorption of moisture from the atmosphere. Samples stored under these conditions for 12 months, have shown no apparent deterioration and in particular no discoloration. If the AF4 solution is being used in an assay, a control experiment should always be carried out. |
| Storage and shelf-life CFM-1 |
| The solution may be stored at room temperature. The cap of the bottle should always be replaced after use. Samples stored under these conditions for 6 months have been found to exhibit little apparent deterioration. |
| Storage and shelf-life CFM-1 plus Antifadent |
| TThe solution may be stored at room temperature. The cap of the bottle should always be replaced after use. Samples stored under these conditions for 6 months have been found to exhibit little apparent deterioration. |
| Storage and shelf-life CFM-2 |
| This glycerol-tris-amine buffered mountant solution, has been specially formulated so as to have a refractive index of ~ 1.52 (at room temperature). It has a pH of ~ 8.5 which is appropriate for fluorescein conjugates. The solution may be stored at room temperature. The cap of the bottle should always be replaced after use. Samples stored under these conditions for 6 months have been found to exhibit little apparent deterioration. |
| Storage and shelf-life CFM-3 |
| This glycerol based mountant solution has been specially formulated so as to have a refractive index of ~ 1.52 (at room temperature) and contains a phenolic type antifadent to retard the bleaching of fluorochromes. The solution is of medium viscosity, is water-white in appearance and has a pH of ~6.5. The cap of the bottle should always be replaced after to use to prevent the ingress of moisture which will lower the refractive index. Solutions should be stored at temperatures between 20 and 22oC. If stored at lower temperatures, crystals may appear in the solution. Warming the solution to between 25 and 28oC will bring about dissolution. |
| Storage and shelf-life AF3 |
| AF3 has an anti-bleaching (antifadent) contained in PBS (phosphate-buffered saline) solution. The solution has a pH of ~10, a refractive index of 1.338 at 20°C and is optically transparent from 300nm into the 750nm. |
| Storage and shelf-life CFPVOH plus AF-100 |
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The CFPVOH is an aqueous solution of poly(vinyl alcohol) and the AF100 is a phosphate-buffered saline solution of an antifadent. Solutions should be made up by mixing 1 part by volume of AF100 with 9 parts by volume of CFPVOH. These solutions should be used within 10 hours as the efficacy of the antifadent reduces with time. With this in mind, it is better to make up the mixture prior to use and not to rely on keeping solutions. The CFPVOH is an aqueous solution of poly(vinyl alcohol) and the AF100 is a phosphate-buffered saline solution of an antifadent. Solutions should be made up by mixing 1 part by volume of AF100 with 9 parts by volume of CFPVOH. These solutions should be used within 10 hours as the efficacy of the antifadent reduces with time. With this in mind, it is better to make up the mixture prior to use and not to rely on keeping solutions. |
