Molecular Probes leads the pack in the development of new fluorescence techniques for live and fixed imaging of biospecimens. After they became a division of Invitrogen, the Molecular Probes line of products continued to improve and innovate. ProLong® Gold Antifade is the perfect example of one product that has been revamped from its predecessor, ProLong® Antifade.
The Gold version of this mounting medium is all about convenience. Its predecessor required mixing of a powder and liquid component, using within a short period of time, discarding the excess, and did not include DAPI. All these convenience problems are solved in the new Gold version which is premixed, can be stored and reused, and comes with optional DAPI. All this convenience costs twice as much, but you don’t waste a drop of it. It is also available in ‘special packaging’. All this means is that the reagent is pre-aliquotted for about $13 more. When comparing this product to the competition’s mounting media, keep in mind that ProLong® Gold is a glycerol-based hard mount media. This means that it cures over 24 hours to a solid state so coverslips do not need to be sealed to the slide with products like nail polish or beeswax. Slides can also be visualized immediately after addition of ProLong® and then stored at room temperature during and after curing. So you don’t have to prewarm the slides to prevent condensation from fogging them between viewings.
When deciding on a mounting medium, besides convenience, the most important factors to consider are how your fluorophore responds to the medium, how long you need to expose your samples to the laser and how long you need to be able to store and reuse the slides. ProLong® works especially well with the Molecular Probes line of Alexa fluorophores and traditional FITC/TX Red/Cy5 fluorophores. It also has no autofluorescence under UV. This makes it superior to the major competitor, Vectashield, which can produce autofluorescence when visualizing FITC or DAPI, and also quenches cyanine dyes like Cy3 and Cy5. ProLong® also works very well in our hands for the preservation of eGFP, eYFP, and eCFP signals. However, ProLong® cannot be used with quantum dots.
How long are you exposing your slides to the laser? If you are exposing your slides to lasers for less than 5 seconds you have many options for mounting media and may not need an antifade reagent at all. However, typical eye scanning of the slide to find the appropriate field of view will take 30 seconds or more. Also, if you are multi-pass imaging, such as is used in confocal imaging, your sample will be exposed for even longer. In this case, an antifade is recommended and may be necessary to preserve the signal during the imaging process. Comparisons in the product manual of ProLong® Gold show that it both enhances and preserves signal for longer periods of time than other comparable mounting media. Vectashield is not included in this particular comparison.
How long do you need to be able to refer back to your samples? ProLong® will keep your signal strong for several months of room temperature storage. Although we have never had a problem with this, to prevent shrinkage of the media and distortion of your samples you can seal the edges of your sample once it cures (after 24 hours) and store the slides at 4°C or 20°C. Also, be aware that unlike aqueous media, the refractive index of curing ProLong® does not stabilize until about 80 hours, so this may change your resolution during this time. These same concerns would apply to other hard mount glycerol-based mounting media. We also find that the long term storage of aqueous and other liquid media is prone to problems with dehydration and distortion due to breaks in the sealant. The hard mount ProLong® does not seem to be as prone to developing sealant leaks in our hands, which is another reason we prefer it to other reagents.
Our lab exclusively uses ProLong® Antifade Gold with or without DAPI. We use this product for mounting of immunofluorescently labeled adherent single cell layers, suspension cells, and cryosectioned tissues. The convenience of use, cleaner storage of slides and recovery of signal after months of storage have made this our product of choice for all immunofluorescence mounting.