ProLong™ Gold Antifade Mountant with DNA Stains DAPI, 10 mL - FAQs

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.

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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.

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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.

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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.

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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.

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The tissue likely had air trapped in it, either before the labeling process or as a result of air-drying. While the tissue is in blocking solution or another wash, we recommend putting it in a vacuum to pull out internalized air. Air-drying is not necessary; just tap off excess buffer and mount the damp tissue with the mountant.

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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.

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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.

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We offer ProLong Coverslip Sealant (Cat. No. P56128) that can be used to seal the edges of the coverslip and is compatible with both curing and non-curing mountant. The sealant is easy to apply and is brushed on after the mountant has cured, for long preservation of slides. The product page can be found here.

When using a hard-curing mountant, such as ProLong Antifade Mountant, make sure the mountant is fully cured before applying ProLong Coverslip Sealant. Since this sealant can seal moisture under the coverslip, it can interfere with the curing process.

When using a non-curing mountant, such as SlowFade Antifade Mountants, sealing can take place immediately after mounting.

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

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

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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').

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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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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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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.

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Some labels, including some antibodies, are of low-enough affinity that they can come off over time during storage of the labeled slides. To slow this off-rate, samples can be post-fixed with formaldehyde for 5-15 min, after the secondary antibody, to cross-link the secondary antibody in place. The sample should also be mounted in a hardening mounting medium, such as ProLong Diamond Antifade Mountant, as the hardening mountant slows diffusion of the secondary antibody. Finally, after the mountant has fully hardened, the slide can be stored cold, preferably at -20 degrees C, to further slow any dissociation.

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Bubbles may be removed by one of two methods:

1. Place the amount of ProLong reagent you wish to use on your sample (plus a little excess) in a microcentrifuge tube. Close the cap and centrifuge this aliquot using a tabletop microcentrifuge (speed from 7, 000 to 13,000 rpm). Bubbles should move to the top and these bubbles may be aspirated using a pipettor/pipette tip.
2. Unscrew the lid of the bottle/vial containing the ProLong reagent to make it loose, but do not remove the lid. Place the entire bottle/vial into a vacuum flask, using a faucet aspirator (faucet T-tube). Apply a vacuum (water running through the faucet) and allow vacuum aspiration to occur from 10 to 20 minutes to degas the mixture.

To avoid the formation of bubbles on a sample or to remove bubbles:

1. Before pipetting the desired amount of ProLong reagent for mounting, set the pipettor for a slight excess volume. When pipetting up the mixture, do not pipette up the complete amount, but lift up the pipette tip from the bottle with the pipettor not yet up to full volume. This prevents the aspiration of bubbles into the pipette tip.
2. Bubbles trapped during application of the coverslip: When placing your coverslip onto your drop of ProLong reagent, place the coverslip at a slight angle then, gently lower the coverslip. If the coverslip is lowered flat onto the sample, or lowered too quickly, bubbles can be trapped.
3. Bubbles trapped in tissue: One problem with tissue sections, particularly cryosections is that air can get trapped within and under the section. Upon mounting, bubbles are not observed but as the mountant hardens, it compresses the sample slightly, forcing air out of the section. This leads to microscopic bubbles forming over the section, trapped within the mountant. To avoid this, degas the tissue sample prior to mounting. Place the sections submerged in buffer or blocking solution, into a vacuum chamber and expose the sample to the vacuum. This will degas the sections and buffer. Remove the sample from this degassed buffer and mount.
4. If ProLong-mounted samples have already cured but have bubbles, you can un-mount your sample by placing the slides into PBS (Coplin jar or a Petri dish filled with PBS). The ProLong reagent will swell and the coverslip will slide off or can be gently removed manually. You can then re-mount with a new aliquot of ProLong reagent.

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DAPI is a very common blue-fluorescent dye for nuclear counterstaining and gives very bright labeling on nuclei in fixed and permeabilized cells and tissues. However, it is considered to be a semi-permeant to impermeant stain and provides inconsistent staining of live cells. Hoechst 33342 dye is cell-permeant and stains with the same binding mechanism and fluorescent color; it is preferred for live-cell imaging and is just as good as DAPI for fixed cell labeling.

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