A non-radioactive in situ hybridization method that does not require RNAse-free conditions
A non-radioactive in situ hybridization method that does not require RNAse-free conditions
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DOI：
10.1016/s0165-0270(98)00123-x
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发表时间：
1998-12
影响因子：
3
通讯作者：
E. Tongiorgi;M. Righi;A. Cattaneo
中科院分区：
文献类型：
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作者：
E. Tongiorgi;M. Righi;A. Cattaneo
本报告描述了一种快速和通用的方法来进行非放射性原位杂交，其中没有杂交步骤在无RNA酶的条件下进行。这项研究表明，原位杂交可以在没有RNA酶的环境中进行，前提是实验期间引入的RNA酶浓度不达到0.1 μg/ml，这是不太可能通过意外污染达到的浓度。此外，提供的证据表明，对RNA酶降解敏感的唯一步骤是预处理，因为在杂交步骤期间由于甲酰胺施加的非常有效的保护作用而不会发生降解。最后，我们的数据表明，内源性RNA酶活性可能很容易通过多聚甲醛固定中和。这种方法的一个特点是牢固的固定，确保完美的组织保存，即使在细胞突起的精细结构水平。该方法允许通过高碘酸钠和硼氢化钠处理均匀的组织渗透，并且可以容易地与二氨基联苯胺免疫组织化学结合用于双标记实验。
This report describes a quick and versatile method to perform non-radioactive in situ hybridization in which none of the hybridization steps are performed under RNAse-free conditions. This study demonstrates that in situ hybridization can be performed without an RNAse-free environment provided that the concentration of RNAse introduced during the experiment does not reach 0.1 μg/ml, a concentration that is unlikely to be achieved through an accidental contamination. Moreover, evidence is provided that the only step sensitive to RNAse degradation is the pretreatment since degradation during the hybridization step can not occur due to a very efficient protective effect exerted by formamide. Finally, our data suggest that endogenous RNAse activity might be readily neutralized through paraformaldehyde fixation. A feature of this method is the strong fixation that ensures a perfect tissue preservation, even at level of the fine structure of the cell processes. The method allows a uniform tissue penetration by sodium periodate and sodium borohydride treatment and can be easily used in combination with diaminobenzidine immunohistochemistry for double labeling experiments.