我用二抗标记我的细胞或组织,然后封片观察。但是标记好像褪色或是随时间而变弱了。我该如何阻止这一现象?
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。
我该怎样去除混合了DAPI的ProLong Antifade Mountant、ProLong Diamond Antifade Mountant和ProLong Diamond Antifade Mountant抗淬灭封片剂中的气泡?
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。
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.
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.
Find additional tips, troubleshooting help, and resources within our Cell Imaging 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.