A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments
A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments
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DOI：
10.1016/s0143-4160(97)90042-7
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发表时间：
1997-03-01
期刊：
影响因子：
4
通讯作者：
Jones, OT
中科院分区：
文献类型：
--
作者：
Tymianski, M;Bernstein, GM;Jones, OT
无法确定非荧光有机钙螯合剂如BAPTA的精确细胞内位置是一个持续存在的问题，这阻碍了对螯合剂在活细胞中的空间或时间动力学进行更详细的分析。类似地，在用荧光指示剂如Fura-2进行生理学实验之后，通常期望将染料保持在细胞内以用于随后通过另外的组织学技术进行分析。基于化学考虑及其在组织固定中的先前使用，我们检查了水溶性试剂1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）作为不同钙螯合剂的潜在固定剂。通过电生理学手段，通过利用抗BAPTA抗体评估固定程度和动力学的新型ELISA，通过负载[C-14]-BAPTA的神经元的放射自显影，以及通过免疫细胞化学和神经元中细胞内BAPTA或钙绿色的成像，证实了EDC而非其他常见固定剂的效用。在浓度> 0.1 mg/ml时，EDC几乎引起> 95%的BAPTA游离酸的瞬时、不可逆固定。在加载有BAPTA/AM酯的海马脑切片中证实了细胞内BAPTA的固定，并且显示出与螯合剂的快速加载和随后的挤出一致的双相动力学。用BAPTA游离酸和荧光黄染料显微注射的神经元的免疫细胞化学显示BAPTA特异性染色，其分布在细胞中类似于伴随的标记染料。EDC的应用还有效地原位固定了BAPTA类似物，例如钙绿色（荧光Ca 2+指示剂），如装载有该指示剂的EDC固定的脑切片的共聚焦成像所示。综上所述，这些数据表明，EDC是一种有效的，廉价的和通用的固定剂的钙螯合剂在不同的细胞。一个合适的固定剂的可用性，现在可以确定这样的螯合剂在光，并可能，电子显微镜水平的分布。EDC的两个重要特征源于其对游离羧基的特异性。第一，选择性地固定螯合剂而不是其AM酯的能力;第二，其作为目前可用的许多其他含羧基螯合剂、染料和pH指示剂的固定剂的巨大潜力。
The inability to determine the precise intracellular location of non-fluorescent organic calcium chelators such as BAPTA is a persistent problem which has precluded much detailed analysis of the chelators' spatial or temporal dynamics in live cells. Similarly, following physiological experiments with fluorescent indicators like Fura-2, it has often been desirable to maintain the dye within the cell for later analysis by additional histological techniques. Based on chemical considerations, and its prior use in tissue fixation, we examined the water soluble reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a potential fixative for diverse calcium chelators. The utility of EDC, but not other common fixatives, was confirmed through electrophysiological means, through a novel ELISA, which exploits anti-BAPTA antibodies to assess the extent and kinetics of fixation; by autoradiography of neurons loaded with [C-14]-BAPTA, and by immunocytochemistry and imaging of intracellular BAPTA or Calcium Green in neurons. At concentrations > 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of > 95% of BAPTA free acid. Fixation of intracellular BAPTA was confirmed in hippocampal brain slices loaded with BAPTA/AM ester, and showed biphasic kinetics consistent with rapid loading and subsequent extrusion of the chelator. Immunocytochemistry on neurons microinjected with BAPTA free acid and the dye Lucifer Yellow showed BAPTA-specific staining which was distributed in the cell similarly to that of the accompanying marker dye. Application of EDC also efficiently fixed in Situ analogs of BAPTA such as Calcium Green (a fluorescent Ca2+ indicator) as shown by confocal imaging of EDC-fixed brain slices loaded with this indicator. Taken together, these data show that EDC is an effective, inexpensive and versatile fixative for calcium chelators in diverse cells. The availability of a suitable fixative now makes it possible to determine the distributions of such chelators at both the light and, possibly, the electron microscope level. Two important features of EDC, arise from its specificity for free carboxyl groups. First, the ability to fix, selectively, the chelators but not their AM esters; and, second, its enormous potential as a fixative for the numerous other carboxyl-containing chelators, dyes and pH indicators currently available.