我希望采用抗淬灭封固剂来封固自己的染料标记细胞,防止染料发生光漂白。你们推荐采用哪种封固剂?
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。
当我拍照时我发现荧光染料的信号淬灭了,我该如何终止这一现象?
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。
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.
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 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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.