Zoeller R T, Fletcher D L, Butnariu O, Lowry C A, Moore F L
Department of Biology, University of Massachusetts, Amherst, USA.
J Histochem Cytochem. 1997 Jul;45(7):1035-41. doi: 10.1177/002215549704500712.
We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3P = S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxynucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.
我们预测，使用35S标记探针进行原位杂交（ISH）后，背景标记的一个重要来源归因于探针的硫代磷酸酯部分[O3P = S]与组织中的二硫化物之间的化学反应。这些共价键会使探针固定在组织中，从而增加背景标记。基于这一观点，我们探索了使用N-乙基马来酰亚胺（NEM）不可逆地烷基化探针的硫代磷酸酯部分和/或烷基化组织中的游离巯基以阻止二硫化物的形成，作为减少背景标记的一种方法。我们报告称，NEM可显著降低35S标记的寡脱氧核苷酸或cRNA探针的背景标记，但不影响特异性标记。我们得出结论，如本文所述，在ISH实验方案中使用NEM可能是研究人员为提高信噪比可考虑的一个额外因素。
