Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues
Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues
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DOI：
10.1078/0344-0338-00192
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发表时间：
2002-01-01
影响因子：
2.8
通讯作者：
Vollmer, E
中科院分区：
文献类型：
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作者：
Goldmann, T;Wiedorn, KH;Vollmer, E
我们报告了HOPE固定（HOPE = Hepes-谷氨酸缓冲液介导的有机溶剂保护效应）用于原位杂交靶向mRNA的标本。为此，开发了一种优化方案，并在HOPE固定的肺标本上重复测试。我们观察到，无论是预处理，在一些实施方案中，预杂交可以使细胞透化，也不需要预杂交来产生信号。脱蜡后，随机引物地高辛标记的探针直接与酵母tRNA杂交在一起，用于阻断非特异性信号。使用与碱性磷酸酶和新品红或NBT/BCIP缀合的抗地高辛抗体作为底物进行检测。通过RT-PCR和适当的阴性对照验证结果。人表面活性蛋白-A和干扰素-γ-诱导蛋白-10的信号在10分钟内迅速发展，伴随着高信号强度，与免疫组织化学中观察到的信号强度相当。通过生物素基-酪胺的信号增强，虽然也给出了合适的结果，但没有导致更高的信号强度，因此不需要与迄今为止测试的探针结合。这些实验使用在适当条件（+4 ℃）下储存长达5年的材料进行。总之，这些初步的结果，获得了新的霍普固定剂，是有前途的，在未来的原位杂交的能力的增强方面。
We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment., permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degreesC) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.