An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative
An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative
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DOI：
10.1016/s0165-0270(96)02204-2
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发表时间：
1997-04-04
影响因子：
3
通讯作者：
Uylings, HBM
中科院分区：
文献类型：
--
作者：
Evers, P;Uylings, HBM
在过去的5年里，微波炉已经成为一种被广泛接受的设备，可以回收被长时间固定的甲醛掩盖的各种抗原。然而，到目前为止，仅用一种方法还不可能对我们感兴趣的所有抗原都获得良好的结果。这项研究为这一问题提供了解决方案。我们测试了Tris缓冲盐水(TBS)的pH 8-10，以了解微波(MW)在高pH溶液中是否对我们特别感兴趣的抗原起作用：MAP-2，非磷酸化部分。神经丝(SMI-32，SMI-311)，神经丝的磷酸化部分(SMI-312)，以及钙结合蛋白Calbindin D28-K，Parvalrin和Calretinin。此外，我们还检查了是否可以使用90摄氏度的较低温度(而不是煮沸组织)来获得同样好的染色结果。这些试验表明，在TBS pH为9.0的全功率(煮沸)下进行微波预处理效果最好。当照射时间延长到30min时，较低温度的预处理对大多数(但不是全部)抗体也有较好的效果。优化的抗原修复方法还改善了神经肽Y(NPY)抗体的染色质量，到目前为止，即使在长时间固定后，似乎也不需要抗原修复。(C)1997年爱思唯尔科学公司。
In the last 5 years the microwave oven has become a widely accepted apparatus for retrieving all kinds of antigens that are masked by prolonged formaldehyde fixation. However, it has so far not been possible to obtain good results for all the antigens we were interested in with just one method. This study offers a solution for this problem. We tested Tris buffered saline (TBS) pH 8-10 in order to see whether a microwave (MW) pretreatment in a solution with a high pH works on the antigens that have our particular interest: MAP-2, non-phosphorylated part of. the neurofilament (SMI-32, SMI-311), phosphorylated part of the neurofilament (SMI-312) and the calcium binding proteins Calbindin D28-K, Parvalbumin and Calretinin. Furthermore we checked whether or not a lower temperature of 90 degrees C (instead of boiling the tissue) could be used to achieve the same good staining results. These tests showed that an MW pretreatment in TBS pH 9.0 at full power (boiling) gave the best results. Pretreatment at a lower temperature gave also good results for most (but not all) antibodies when the irradiation time was prolonged to 30 min. The optimal antigen retrieval method also improved the quality of staining with an antibody against neuropeptide Y (NPY), for which, until now, no antigen retrieval appeared to be necessary, even after a long duration of fixation. (C) 1997 Elsevier Science B.V.