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首页> 外文期刊>European Journal of Histochemistry >New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining
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New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4',6-diamidino-2-phenylindole (DAPI)-staining

机译：通过4'，6-二mid基-2-苯基吲哚（DAPI）染色对无机多磷酸盐存储原位显微镜进行可视化和定量分析的新见解

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摘要

Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4',6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multi-photon microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.
机译：无机多磷酸盐（PolyP）是一种生物聚合物，在原核生物和真核生物的细胞生理中均起着重要作用。在用于PolyP定位和定量的可用方法中，基于4'，6-diamidino-2-phenylindole（DAPI）的测定法已用于可视化PolyP丰富的细胞器。由于固定后DAPI对不同隔室的通透性和/或PolyP保留的差异，尚未建立DAPI-PolyP染色的通用协议。在这里，我们测试了具有不同DAPI渗透水平的一系列样品中DAPI-PolyP检测的不同方案，包括亚细胞级分，自由活动细胞和固定组织的冰冻切片。富含PolyP的细胞器的亚细胞级分产生DAPI-PolyP荧光，尽管具有复杂外层的细胞通常需要更长的孵育时间，先前的醛固定和/或去污剂渗透性。通过多光子显微镜分析，在OCT包埋组织的冷冻切片中也检测到了DAPI-PolyP。此外，对DAPI染色级分的半定量荧光分析表明，PolyP动员的方式与使用基于酶的定量方案所证明的方式相似。两者合计，我们的结果支持使用DAPI进行PolyP可视化和定量分析，尽管建议采用特定步骤作为DAPI和PolyP渗透度不同的生物样品中DAPI-PolyP染色的一般指南。

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