A photoreactive fluorescent marker for identifying eosinophils and their cytoplasmic granules in tissues
A photoreactive fluorescent marker for identifying eosinophils and their cytoplasmic granules in tissues
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DOI：
10.1177/002215540305100214
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发表时间：
2003-02-01
影响因子：
3.2
通讯作者：
Beuving, LJ
中科院分区：
文献类型：
--
作者：
Eversole, RR;Mackenzie, CD;Beuving, LJ
在这里，我们描述了一个简单的组织化学技术，提供了一种改进的方法来识别组织中的嗜酸性粒细胞成分，通过形成光反应复合物，产生稳定的荧光发射。该方法容易与6-60 μ m厚的组织学组织切片一起工作，其固定在中性缓冲福尔马林（NBF）中，并且与类似地固定和未固定的细胞悬液一起工作。当暴露于来自100-W汞灯源（510-590 nm）的宽带激发光谱以及来自氩激光（488 nm）和UV-可见激光（514 nm）的单波长激发时，嗜酸性粒细胞特异性颗粒产生深红色（>605 nm）荧光发射。荧光团-颗粒复合物发射在连续光激发的第一分钟期间强度增加，然后保持稳定（>10分钟）。与这些组织相关的所有非特异性自发荧光现象在第一分钟内被光漂白，包括未形成光反应性复合物的背景Biebrich猩红结合区域（即，胶原），表明环境对荧光团的影响。该技术允许在比通常允许的标准荧光标记物更长的时间段内观察嗜酸性粒细胞颗粒。因此，技术，如共聚焦显微镜可以利用到最大程度，提供更详细的信息的嗜酸性粒细胞的细胞质内容物的位置和分布。
Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photo-reactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6-60 mum thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510-590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.