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首页> 外文期刊>Journal of Neuroscience Methods >An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative.
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An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative.

机译:一种最佳的抗原回收方法,适用于在甲醛固定剂中保存数年的人脑组织上的不同抗体。

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摘要

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.
机译:在最近的5年中,微波炉已成为一种广泛接受的设备,用于回收被长时间的甲醛固定所掩盖的所有抗原。但是,到目前为止,仅用一种方法就不可能获得我们感兴趣的所有抗原的良好结果。这项研究为该问题提供了解决方案。我们测试了pHs 8-10的Tris缓冲盐水(TBS),以查看在高pH值溶液中进行微波(MW)预处理是否对我们特别感兴趣的抗原起作用:MAP-2,其非磷酸化部分神经丝(SMI-32,SMI-311),神经丝的磷酸化部分(SMI-312)和钙结合蛋白钙结合蛋白D28-K,小白蛋白和钙黄蛋白。此外,我们检查了是否可以使用90摄氏度的较低温度(而不是使组织沸腾)来获得相同的良好染色效果。这些测试表明,在TBS pH 9.0下以全功率(沸腾)进行MW预处理可获得最佳结果。当辐照时间延长至30分钟时,对于大多数(但不是全部)抗体,在较低的温度下进行预处理也可获得良好的结果。最佳的抗原回收方法还提高了抗神经肽Y(NPY)抗体的染色质量,到目前为止,即使经过长时间的固定,似乎也没有必要进行抗原回收。

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