ProLong Antifade Kit From Molecular Probes (Invitrogen)

“Last but not least”. This saying perfectly describes the significance of slide mounting during immunohistochemistry, especially using an immunofluorescent detection system. Fluorescent signal tends to fade away with time, thus the use of a proper mounting medium, which can sustain fluorescence, is essential. Molecular Probes (now part of Invitrogen), a company dedicated to the production of fluorescence detection reagents, addressed that issue by developing a two-component antifade mounting medium: ProLong Antifade Kit. The ProLong Antifade Kit not only allows you to spend more time acquiring images during a given session, but also prolongs the life of the fluorescence in your samples, thus allowing you to come back to them after months.

I have successfully used the ProLong Antifade Kit for single-, double-, and triple- fluorescent staining, using a combination of Alexa-based fluorescent dyes with and without DAPI, determining the expression of my proteins of interest in primary neuronal cell cultures, HEK cells and tissue slices. To get the most out of the ProLong Antifade Kit, it is important to remember a few tricks. The kit consists of 2 components: Component A is the antifade reagent, a solid powder aliquoted into 20 vials; Component B is the polymer-based, viscous mounting medium. The kit is stored in -20ºC, but you need to warm up Component B to room temperature prior to mixing it with Component A; thus, don’t forget to take it out from the freezer ~ 30 min in advance. Component A is a solid pellet at the bottom of the vial, thus I would recommend breaking it apart (e.g. with a glass rod) before adding Component B to it. Additionally, Component B is very viscous, thus to mix it well with the other component you should vortex it for at least 2 minutes (which may feel extremely long, but is essential). Vortexing will introduce a lot of bubbles to the medium, so give it a quick spin and suck the medium up into an eye dropper a few minutes prior to putting it on slides – this will give time for the air bubbles to go to the top of your medium in the eye dropper and prevent them from getting onto the slides. Resist the temptation of putting too much medium on your slides - ProLong is very efficient, one drop per a single square coverslip will suffice. Moreover, too much medium will extend the polymerization time and can cause the appearance of undesirable, brown precipitates - products of oxidation. This is important because, due to the use of 1 ml of Component B per each vial of Component A, you will always have an excess of your mounting medium and will probably want to use it all up. Unfortunately, you can not save it for later, and the excess need to be tossed away. This is probably the only drawback of this product. Next, let your medium harden in a dark drawer overnight and seal the slides with a nail polish. Finally, don’t forget to store your slides in a fridge or a freezer to extend life of the slides.

In summary, the ProLong Antifade Kit is a great mounting medium in immuno-fluorescence application, and I recommend it without hesitance.