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  5. Molecular Probes™ ProLong™ Diamond Antifade Mountant

Molecular Probes™ ProLong™ Diamond Antifade Mountant

Product Code. 15372192
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Product Code. 15372192 Supplier Molecular Probes™ Supplier No. P36965

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Description

When sample thickness and imaging depth is not of concern, ProLong Diamond Antifade Mountant provides superior photobleach protection for Alexa Fluor dyes and fluorescent proteins allowing long term imaging experiments without loss of signal.

ProLong Diamond Antifade Mountant is a glycerol-based liquid mountant applied directly to fluorescently labeled cells or tissue samples. ProLong Diamond mountant is designed to provide unparalleled antifade protection across the entire visual and IR spectrums. You can use ProLong Diamond mountant with almost any fluorescent dye or fluorescent protein (e.g., GFP, RFP, mCherry) to achieve the brightest signal and lowest background, for bright, high-definition fluorescent images.

ProLong Diamond Antifade Mountant comes mixed and ready-to-use in a 2 mL dropper bottle or 10 mL bottle. Just apply a drop to the sample, add a coverslip, cure, and image. ProLong Diamond mountant does not discolor or shrink when cured, enabling high-quality fluorescent images months after mounting your sample. ProLong Diamond mountant will cure to an RI of 1.47.

Get photobleaching protection across the entire visible and IR spectrums
ProLong Diamond Antifade Mountant’s ability to provide brighter signal, against low background, helps ensure that you get high-resolution images. ProLong Diamond provides superior protection against photobleaching and will not quench your fluorophores. It can be used with an array of fluorescent dyes, including Alexa Fluor dyes, FITC, TRTC, Texas Red, Cy3, and Cy5 or fluorescent proteins. For immediate viewing of the sample without curing, SlowFade Diamond Antifade Mountant is optimal.

Archiving fluorescently labeled samples with excellent RI
ProLong Diamond mountant is the ideal option for archiving samples with fluorescent dyes or fluorescent proteins. Please note that ProLong Glass mountants are recommended for samples > 10 microns and all immersion oil imaging applications. It is also ideal for preserving signal strength during prolonged imaging sessions, such as repeated imaging under confocal laser scanning illumination, or with super resolution microscopy. Additionally, the curing property of ProLong Diamond means you do not need to use traditional acrylic resins (i.e., fingernail polish), which can damage microscope objectives if it comes into contact with them. The refractive index determines how much the path of light is bent, or refracted, when entering a material. In microscopy, refractive index differences between specimen and mounting medium should be minimized and can be reduced by using mountants with a higher RI. Optimizing the RI of your experiment will increase the resolution to provide better quality images. Depending on your sample type, thickness, imaging depth and/or staining reagents (e.g. fluorescent proteins or dyes) and whether you need a curing or non-curing mountant, choosing the right mounting media will impact image resolution and should be strongly considered for optimal experimental design.

TRUSTED_SUSTAINABILITY

Specifications

Quantity 2 mL
Product Type Antifade Mountant
Content And Storage Contains 1 dropper bottle contaning 2 mL reagent.

• Store at 2-8°C.
• Protect from light.
• Stable for at least 6 months after receiving.
Shipping Condition Approved for shipment at Room Temperature or on Wet Ice
Product Line ProLong
Reagent Type Mounting Solution, Antifade Solution
Volume (Metric) 2 mL
Description ProLong™ Diamond Antifade Mountant, 1 x 2 mL

Frequently Asked Questions (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.

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.

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.
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 (37oC) 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.
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.

I mounted my antibody-labeled brain cryosection in either ProLong Antifade Mountant, ProLong Gold, ProLong Diamond or ProLong Glass Antifade Mountant. It looked good when first mounted, but after curing, it had tiny bubbles all over the tissue under the coverslip. The sample was air-dried prior to mounting. What happened and what can be done to remove the bubbles?

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.
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.
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.
I do not have epoxy or VALAP to seal the coverslip. Do you offer an alternative coverslip sealant?

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.

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

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

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.

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').

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.
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.
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.
I labeled my cells or tissues with a secondary antibody, and then archived my slides. But the label seems to be fading or coming off over time. What can I do to prevent this?

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.
I want to mount my dye-labeled cells in an antifade mounting medium to keep the dyes from photobleaching. Which mounting medium do you recommend?

As dyes are illuminated for imaging, they will fade, or “photobleach”, leading to unwanted dimming and lower detection efficiency over time. An antifade mounting medium can greatly reduce photobleaching. If you wish to label live cells, use of ProLong Live Antifade Reagent is helpful. If you wish to mount fixed cells after labeling, and then image immediately and then discard, SlowFade Diamond Antifade Mountant stays liquid but has good refractive index. If you wish to mount your sample and then archive the slides, ProLong Diamond Antifade Mountant will harden to a better refractive index and allow for archiving of the sample for up to several weeks, or even months. Unlike other antifade mounting media, these work well with fluorescent proteins for immediate viewing (archiving fluorescent proteins is not possible), and they are packaged with or without DAPI. More information on these can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/mounting-medium-antifades/prolong-gold-antifade.html).
How do I remove bubbles from the vials of either ProLong Antifade Mountant, ProLong Gold, ProLong Diamond or ProLong Glass Antifade Mountants or ProLong-mounted samples?

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.

My fluorescent dye signal is fading as I image it. What can I do to stop this?

All fluorescent dyes will fade, or “photobleach”, to at least some extent when exposed to strong light at the wavelengths they absorb. Here are some causes for photobleaching and ways to fix the problem:

1) Cause of photobleacing - Generation of free radicals and singlet oxygen
Remedy - i) Use an antifade reagent, which has antioxidants and free radical scavengers:

ii) For live-cell imaging of fluorescent dyes and proteins, we recommend ProLong Live Antifade Reagent which can be added to the cell media or buffer. ProLong Live Antifade Reagent can significantly increase the stability over time for reagents as well as fluorescent proteins, like GFP, without affecting cell health, for up to 24 hours.

iii) For immediate analysis and short-term storage of fixed samples, we recommend SlowFade Diamond Antifade Mountant (it is a liquid mountant and can be used for immediate viewing and then disposal of the sample within a day).

iv) For long-term analysis of Alexa Fluor dyes in fixed samples, we recommend a mountant that hardens, such as ProLong Diamond Antifade Mountant. The harrdening of the mountant also slows diffusion of free radicals).

v) For long-term analysis of all dyes and fluorescent proteins in fixed samples, we recommend ProLong Diamond Antifade Mountant, suitable for archiving slides.

2) Cause of photobleaching - Dye is particularly sensitive to structural modification upon exposure to light.
Remedy - i) Choose a more photostable dye, such as many of our Alexa Fluor dyes.

3) Cause of photobleaching - Intense Illlumination
Remedy - i) Reduce light exposure, for example by reducing laser power or using neutral density filters.

ii) Minimize the viewing time of labeled sample, and close shutter when not viewing.

iii) Use an objective with a lower numerical aperture, such as a lower-power objective.

You can find more information on choosing an antifade reagent on the link below http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/mounting-medium-antifades.html.

Safety and Handling

Product Identifier
  • ProLong Diamond Antifade Mountant
 Signal Word
  • Warning
 Hazard Category
  • Skin corrosion/irritation Category 2

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