Dagerlind A, Friberg K, Bean A J, Hökfelt T
Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden.
Histochemistry. 1992 Aug;98(1):39-49. doi: 10.1007/BF00716936.
In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-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 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 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.
在本研究中，使用35S标记和碱性磷酸酶偶联探针分析了原位杂交组织化学（ISHH）的一些实验参数，以建立一种可重复的双重标记方法。我们比较了完全不进行组织固定与经福尔马林浸泡固定的组织切片。此外，还评估了二硫苏糖醇在将放射性标记探针和非放射性探针组合用于单个组织切片时以及单独使用探针时的效果。对于放射性标记探针和碱性磷酸酶偶联探针，未固定组织的杂交产生更强的特异性标记和更低的背景。未固定组织杂交后，在光学显微镜水平未见组织保存受损。使用35S标记探针时，高浓度（200 mM）的二硫苏糖醇强烈抑制背景，而在非放射性方法中，只有在非常低的二硫苏糖醇浓度（小于1 mM）下才能实现碱性磷酸酶标记。这种不相容性导致了一种使用未固定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的二硫苏糖醇，第二步使用不含二硫苏糖醇的碱性磷酸酶偶联探针。
