Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections
Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections
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DOI：
10.14670/hh-25.1017
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发表时间：
2010-08-01
影响因子：
2
通讯作者：
Neder, L.
中科院分区：
文献类型：
--
作者：
Oliveira, V. C.;Carrara, R. C. V.;Neder, L.
自体荧光的干扰是原位杂交诊断检测中免疫荧光分析的主要问题之一。我们提出了一种有用的技术，在不影响组织完整性或脑切片中直接免疫荧光信号的情况下，减少了自发荧光背景。在福尔马林固定石蜡包埋和冰冻人脑组织切片上，采用氨水/乙醇、70%乙醇苏丹黑B(SBB)、紫外光漂白以及两者的不同组合等六种不同的方法，我们发现0.1%的SBB在70%乙醇中的组织处理是最好的方法来减少/消除组织的自发荧光和背景，同时保留特定的荧光杂交信号。这种策略是一种可行的、不耗时的方法，它在组织自体荧光的总体减少和特定荧光标记的维护之间提供了合理的折衷。
Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.