Epi-fluorescent and confocal imaging techniques are key tools in providing visual information in research. High power confocal laser systems have made it easier to look at multiple antigens in a sample. The drawback to these systems, however, is the possibility of photobleaching, which limits the number of confocal scans. The possibility of bleaching makes it difficult to experiment with different exposure conditions in order to find the most favorable image. There are several antifade reagents on the market that can reduce bleaching of a sample; however, it is important that one chooses the right reagent for the desired application.
Along with photobleaching, other concerns when working with immunofluorescence include coverslips that move around on the slide, getting aqueous mounting media on very expensive microscope objectives, and the preservation of signal over an extended period of time. Molecular Probes (Invitrogen) has made it easier to choose the best antifade reagent. Their product, ProLong® Gold, is a ready-to-use antifade reagent that significantly reduces bleaching and allows experiments to be saved for months after mounting. This antifade reagent cures to permanently fix coverslips in place, which prevents coverslips from moving around and destroying objectives with media.
We use the reagent containing DAPI for a quick nuclear counterstain. There is a product available without DAPI that works just as well for mounting. To extend the use of the product, we find it convenient to make 500 microliter aliquots and store them at -20°C upon arrival. Prior to application, we thaw the reagent to room temperature and spin it down to remove bubbles. The slide is then blotted on a paper towel to remove excess buffer. One drop of antifade reagent is placed on each sample and subsequently covered with a coverslip. The slides are then left in the dark at room temperature for 24 hours to allow polymerization. The slides can then be sealed to avoid shrinkage from the curing process. For long term storage, the slides are placed at -20°C and the signal remains viable for months.
Due to the viscosity of the reagent, it is very important that the reagent is free of bubbles. However, since only one drop is required per section, as long as attention is given to that drop being bubble-free, the curing process will result in excellent conditions for imaging. It is also very important to allow the slide to completely cure in order for the reagent to perform at its best in reducing bleaching.
ProLong® Gold works very well with a wide range of secondary fluorophores and is a helpful reagent for multicolor applications. We routinely run immunofluorescence experiments on cultured cells as well as mouse, rat, and human tissue. Triple labeling is in high demand and I frequently use antibodies of different host species in combination with Alexa 488, Alexa 555, and Alexa 647. It is very convenient to mount with ProLong® Gold with DAPI for the additional fourth color channel displaying the DAPI-stained nuclei. The hard-setting property of the reagent allows us to place the slides on our inverted confocal microscope. Several trial scans can be performed prior to final image acquisition without the worry of photobleaching.
This antifade reagent allows one to obtain the best images possible from an immunofluorescence experiment. The greatest feature is that it enables the researcher to revisit the slides months after staining in order to answer questions that may surface over the course of the project. The customer service of the company itself is impeccable and they provide a very thorough product specification sheet with each shipment.
Surita Banwait
Research Associate II
Buck Institute for Age Research
Morphology & Imaging Core