SENSITIVE MESSENGER-RNA DETECTION USING UNFIXED TISSUE - COMBINED RADIOACTIVE AND NONRADIOACTIVE INSITU HYBRIDIZATION HISTOCHEMISTRY
SENSITIVE MESSENGER-RNA DETECTION USING UNFIXED TISSUE - COMBINED RADIOACTIVE AND NONRADIOACTIVE INSITU HYBRIDIZATION HISTOCHEMISTRY
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DOI：
10.1007/bf00716936
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发表时间：
1992-08-01
期刊：
影响因子：
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通讯作者：
HOKFELT, T
中科院分区：
文献类型：
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作者：
DAGERLIND, A;FRIBERG, K;HOKFELT, T
在本研究中，使用 S-35 标记和碱性磷酸酶缀合探针分析了原位杂交组织化学 (ISHH) 的一些实验参数，以开发可重复的双标记程序。我们将完全排除组织固定与通过福尔马林浸泡固定的组织切片进行了比较。此外，当在单个组织切片上组合放射性标记和非放射性探针时以及当探针单独使用时，评估了二硫苏糖醇的效果。未固定组织的杂交导致放射性标记探针和碱性磷酸酶缀合探针的特异性标记更强，背景更低。未固定的组织杂交后，在光学显微镜水平上没有看到组织保存的损失。当使用 S-35 标记的探针时，高浓度 (200 mM) 的二硫苏糖醇强烈抑制背景，而在非放射性过程中，碱性磷酸酶标记只能在非常低的二硫苏糖醇浓度 (< 1 mM) 下实现。这种不相容性导致使用未固定的组织切片和顺序杂交程序的方案，第一步使用放射性标记探针和高浓度二硫苏糖醇，第二步使用不含二硫苏糖醇的碱性磷酸酶缀合探针。
In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using S-35-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using S-35-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (< 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.