ProLong™ Gold Antifade Mountant, 2 mL - FAQs

What is the difference between ProLong and SlowFade antifade reagents?

Our ProLong antifade reagents dispense as a liquid that will solidify upon the evaporation of water. SlowFade antifade reagents remain liquid. If you are going to image right away and then dispose of your sample, you do not need a mountant that hardens, such as the SlowFade reagents. If you wish to archive your slide for more than a day, you will want a mounting medium that hardens (or “cures”). This hardening will limit the off-rates of various dye-conjugated antibodies and provides a better refractive index. Also, there will be a lower diffusion rate of free radicals, limiting photobleaching.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

If I use ProLong Gold Antifade Mountant to mount my slides, should I seal the edges?

ProLong Gold Antifade Mountant hardens overnight at room temperature. For short-term storage (a couple of weeks) you do not need to seal the edges of the coverslip, and the sample should be stable. Beyond that time, though, some dye conjugates will have an off-rate into the medium, so cold storage is recommended. Sealing the edges will prevent long-term discoloration (golden color) from developing around the edges of the coverslip as the anti-oxidants oxidize.

The edges may be sealed with melted paraffin, VALAP (1:1:1 vaseline, lanolin, paraffin) or epoxy glue. Nail polish is not recommended as various components of nail polish may diffuse into the mountant and quench fluorescent dyes.

Find additional tips, troubleshooting help, and resources within our Cell Imaging Support Center.

Some antifade mounting media stay as liquid whereas others harden. What is the benefit of having one that hardens?

If you are going to image right away and then dispose of your sample, you probably want a mountant that does not harden. If you wish to archive your slide for more than a day, you want a mountant that hardens (or "cures"). This hardening will slow or prevent off-rate of your dye or conjugate and often produces a better refractive index. Secondary sealing is usually not necessary. Also there will be lower diffusion of free radicals, thus limiting photobleaching.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I mounted my cells in ProLong antifade mounting medium, but now I want to go back and re-label them. Is there a way I can unmount the coverslip after it has cured (hardened)?

Yes. Put the slide in a Coplin jar or beaker filled with warm (37^oC) PBS buffer and let it sit, no agitation is required. The hardened ProLong mountant will swell and may slide off or be easily dislodged. If cells are adherent to the coverslip, make certain the coverslip side containing the cell or tissue sample does not land face down in the container or become scratched upon handling. Remove the coverslip, wash a couple of times, and proceed with re-staining and re-mounting in new ProLong mountant.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I am using ProLong antifade mounting medium. Do I need to let it cure before imaging? Do I need to seal the edges of the coverslip?

You can image before it cures (hardens), and it will still slow photobleaching, but you have to let it cure overnight to get the best refractive index (resolution). There is no need to seal the edges. In fact, if you seal before it cures, it won't cure correctly. If you are archiving the slide for more than a month, though, seal the edges with resin, paraffin or VALAP (1:1:1 vaseline, lanolin, paraffin) after it cures or there may be slight discoloration along the edges over time.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

My dye is photobleaching too fast with confocal imaging. What can I do?

If this is a fixed-cell system, use of an antifade mounting medium, such as ProLong Diamond. You can also use a more photostable dye, such as the Alexa Fluor dyes. For confocal imaging, you can reduce the illumination on any given dye by decreasing laser power, reducing dwell time or average rastering, or increasing scan speed, though these options may also reduce your resolution. You can also shutter the light source whenever you do not need to scan or look at the sample.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I am labeling my paraffin sections with a primary and secondary antibody. We know the primary antibody works by using colorimetric detection, but when using a fluorescent secondary antibody, such as Alexa Fluor 488 goat anti-mouse antibody, it is very dim. I am mounting in CytoSeal Mounting Medium.

A potential problem here is that Cytoseal Mounting Medium quenches many dyes to a large extent. Alexa Fluor 488 is one of those, and may be quenched by as much as 60%. Also, Cytoseal Mounting Medium does not have an antifade component to reduce photobleaching. We recommend using an aqueous mounting medium instead, such as ProLong Diamond Antifade Mountant, which does not quench the dye and includes an antifade.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

What do ProLong Gold, ProLong Diamond and ProLong Glass antifade mountant reagents contain?

They are composed of proprietary antifades and polymers in aqueous buffers.

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Can I use ProLong Gold, ProLong Diamond, or ProLong Glass mounting medium in a multi-well plate?

No. If the sample cannot be sealed with a coverslip, we recommend the use of SlowFade antifade mountant.

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What can affect sample curing of ProLong Gold-, ProLong Diamond-, and ProLong Glass-mounted samples?

Our ProLong Gold, ProLong Diamond, and ProLong Glass mounting media harden ('cure') by the evaporation of water. Placing the mounted samples in a humid or refrigerated environment would slow down the evaporation process ('curing').

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Are ProLong, ProLong Gold, ProLong Diamond, and ProLong Glass reagents compatible with DyLight dye-conjugated antibodies and streptavidin and with other organic dyes?

We have not tested ProLong, ProLong Gold, ProLong Diamond, or ProLong Glass on DyLight dye-conjugated proteins or with other organic dyes, but it is likely that they will be compatible.

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Can I use any of the ProLong, ProLong Gold, ProLong Diamond, or ProLong Glass reagents on live samples?

No. ProLong, ProLong Gold, ProLong Diamond, and ProLong Glass reagents are intended for use on fixed or fixed/permeabilized samples. Only ProLong Live Antifade Reagent (Cat Nos. P36974 and P36975) is intended for use with live cells.

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What happens when ProLong, ProLong Gold, ProLong Diamond, or ProLong Glass antifade mountant is curing? Is there a chemical reaction?

The evaporation of water is the “curing” process for Prolong, ProLong Gold, ProLong Diamond, and ProLong Glass antifade mountant. The evaporation of water causes the polymer to harden. There is no chemical reaction. Please note that there is no curing/hardening for any of the SlowFade reagents or ProLong Live products.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.