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。
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.
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.