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View additional product information for ProLong™ Diamond Antifade Mountant - FAQs (P36970, P36961, P36965)
23 product FAQs found
包括抗体在内的一些标记亲和力较低，它们会在标记载玻片的储存期间随时间而变弱。为了减缓这种解离速率，可以在加入二抗后将样品用甲醛5-15分钟固定，，用交联的方法将二抗固定交联。也可以用固化封固剂（如ProLong Diamond Antifade Mountant）封固样品，因为固化封固剂可以减缓二抗的扩散。最后，封固剂完全固化之后载玻片应低温保存（最好是-20 ℃），从而进一步减缓解离。
染料需要发光成像，在此过程中它们会淬灭或“光漂白”，导致荧光不断变暗、检测效率不断降低。抗淬灭封固剂可以大幅减少光漂白现象。如果您想标记活细胞，可使用ProLong Live抗淬灭试剂。如果您想标记后立即封固细胞，然后立即成像并丢弃样品，可采用SlowFade Diamond 抗淬灭封固剂，因为其可以保持液态并有良好的折射率。如果您想封固样品后存档样品，ProLong Diamond 抗淬灭封固剂会固化以获得更好的折射率，且可存档样品长达数周乃至数月。与其他抗淬灭封固剂不同，此类产品非常适合对荧光蛋白质进行即时成像（无法进行荧光蛋白质存档），并且有含或不含DAPI两种包装规格。更多信息请见此处。
有以下两种方法来去除气泡：
1.吸取少量你们要使用的ProLong 抗淬灭封片剂（少许过量）于离心管中。盖上盖子并置于离心机中离心（转速7, 000到13,000 rpm）。此时气泡会移动到样品顶部，通过移液枪/枪头即可移去气泡。
2.拧松ProLong 抗淬灭封片剂的瓶盖，但不要打开。将整瓶/管放入真空瓶中，使用抽气龙头（抽气龙头T型管）。打开真空（水随龙头流走）并对混合液抽真空10到20分钟。
为了避免在样品中形成气泡或去除气泡：
1.吸取适量的ProLong 抗淬灭封片剂封片之前可以将吸取体积调高一点。吹打混合液时不要把全部液体都吸上来，枪和枪头向上离开瓶子的时候体积不要满。这样做可以防止气泡进入枪头。
2.使用盖玻片时出现气泡：当把盖玻片放于ProLong 抗淬灭封片剂上封固时，将盖玻片抬起一个小角度后再轻轻放下。如果盖玻片离样品平面过近或是放下过快，都有可能出现气泡。
3.组织中出现气泡：组织切片，尤其是冰冻切片的一个问题是，空气往往会困于切片中或切片下。在封固时没有发现气泡，但是随着封片剂变硬，对样品轻微的挤压就会放出其中的空气。这使得切片中形成部分微小气泡，封于封固剂中。为了避免这种情况，封固之前先进行脱气。将切片浸没在缓冲液或封闭液中，然后放入真空泵上抽真空。这样就会把切片上的空气和缓冲液抽去。将切片从脱气缓冲液中移出然后封固。
4.如果ProLong抗淬灭封片剂封固样品时快要凝固但是出现了气泡，你们可以把玻片放入PBS中（装有PBS的Coplin jar或Petri dish）。ProLong抗淬灭封片剂会膨胀而盖玻片会滑落，或者你们也可以用手轻轻移开盖玻片。你们可以用新的小份的ProLong 抗淬灭封片剂重新封固。
当它们暴露在吸收波长强光下时，所有的荧光染料都会在一定程度上出现褪色或“光漂白”现象。这里列有一些光漂白的原因以及解决方案：
1）光漂白的原因：自由基和单线态氧的产生
可能的解决方案：使用具有抗氧化剂和自由基清除剂的抗褪色剂：
i. 对于荧光染料和蛋白质的活细胞成像，我们建议使用ProLong Live Antifade Reagent，该试剂可以加入到细胞培养基或缓冲液中。ProLong Live Antifade Reagent可以在24小时内不影响细胞的健康的前提下显著增加试剂和荧光蛋白，如GFP的稳定性。
ii. 对于直接分析与短期储存的固定样品，我们建议使用SlowFade Diamond Antifade Mountant（该试剂保持液态并能即看即用，然后在一天内丢弃样品）。
iii. 对于长期分析Alexa Fluor染料的固定样品，我们推荐固化的封固剂如ProLong Diamond Antifade Mountant（其可减慢自由基运动）。
iv. -对于长期分析所有染料和荧光蛋白的固定样品，我们推荐ProLong Diamond Antifade Mountant（固化后可以保存载玻片）。
2) 光漂白的原因：染料对褪色尤为敏感
可能的解决方案：
i. 选择更耐光染料，比如我们的Alexa Fluor染料
ii.您可以使用复染剂选择和设置像场，然后切换到目标染色加以成像。
3) 光漂白的原因：强光照射
可能的解决方案：
i. 减少曝光，例如降低激光功率或使用中性密度滤光片。
ii. 观察标记样品的时间尽可能的短，并在不观看时关闭遮板。
iii. 使用具有较低数值孔径的物镜，如低功耗物镜。
这里（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/mounting-medium-antifades.html?icid=fr-antifade-main）是一个很好的抗淬灭剂选择指南。
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.
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.
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.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
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.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.