ProLong® Gold antifade reagent is a ready-to-use mounting medium intended for use with fluorescent imaging techniques. To prepare your sample, you only have to put a drop of this reagent onto your sample and cover it with a coverslip. You should do this at least one day before observation with a confocal microscope. That is all: the sample can be stored for weeks or more. (We had to repeat confocal observations on samples that had been stored for several months at -28°C.)
This medium is available in either a single 10 ml bottle or five 2 ml bottles (“special packaging”); the latter is far more convenient, as these small bottles also function as pipettes. Using the bottle as a pipette allows you to avoid the difficulties of handling this viscous liquid. Without the small pipette-bottles, the reagent tends to transform into multiple bubbles, making mounting very difficult. The 10 ml packaging is not only inappropriate for dispensing the reagent, but it also requires multiple freeze/thaw cycles, depending on the frequency of your experiments, since it must be stored at -20°C. In addition, the time for thawing must be taken into account in timing your experiments.
We use ProLong® Gold antifade reagent as mounting medium for frozen sections of animal tissues in multiple-label immunofluorescent staining. We use different combinations of antibodies conjugated to fluorescent labels, such as Alexa 488, FITC, TRITC, Cy5, Alexa 647, or fluorescent dyes (Red, Green Synaptracers, fluorescent phalloidin dyes, etc.). We have always been satisfied with this product when observing our samples with argon, helium-neon green and helium-neon red lasers. Indeed, ProLong® Gold is much more effective than other mediums which we have used in the past. ProLong® Gold really diminishes quenching of the fluorescent signal and thus, allows higher zoom, higher resolution and numerous scans which are necessary for signal averaging and obtaining convincing confocal data.
It is worth noting that this reagent can take more than the stated 24 hours to polymerize completely, probably depending on the quantity of water remaining on the sections. Nevertheless, if you really have your back against the wall, you can visualize your sample with a confocal microscope even if the ProLong® Gold is not fully polymerized. The only risk you take when using unpolymerized reagent is not being able to focus properly, however, the reagent will still reduce bleaching considerably.
The only drawback of this product is its price. To my knowledge, it is about five times more expensive than other mounting mediums (Gel Mount™, for example, from Sigma-Aldrich), but if you want to be fully satisfied with your sample preparation, especially when the fluorescent signal needs to be quantitatively compared between different samples, you will not regret this choice. In conclusion, this is an outstanding product that I would recommend to others without hesitation.
Alexandra Khomitch-Baud, Ph.D.
Scientist
Biovays
Cellular Imaging Department