Tymianski M, Bernstein G M, Abdel-Hamid K M, Sattler R, Velumian A, Carlen P L, Razavi H, Jones O T
Playfair Neuroscience Unit, Toronto Hospital Research Institute, Toronto Western Hospital, Ontario, Canada.
Cell Calcium. 1997 Mar;21(3):175-83. doi: 10.1016/s0143-4160(97)90042-7.
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 [14C]-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.
无法确定诸如BAPTA等非荧光有机钙螯合剂在细胞内的确切位置一直是个问题，这使得人们无法对螯合剂在活细胞中的空间或时间动态进行详细分析。同样，在用像Fura-2这样的荧光指示剂进行生理实验后，人们常常希望将染料保留在细胞内，以便随后通过其他组织学技术进行分析。基于化学方面的考虑以及其在组织固定中的先前应用，我们研究了水溶性试剂1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）作为多种钙螯合剂的潜在固定剂。通过电生理方法、一种利用抗BAPTA抗体来评估固定程度和动力学的新型酶联免疫吸附测定（ELISA）、对加载了[14C]-BAPTA的神经元进行放射自显影以及对神经元内的BAPTA或钙绿进行免疫细胞化学和成像，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几乎能瞬间、不可逆地固定>95%的BAPTA游离酸。在加载了BAPTA/AM酯的海马脑片中证实了细胞内BAPTA的固定，并且显示出与螯合剂快速加载和随后挤出相一致的双相动力学。对微注射了BAPTA游离酸和染料路西法黄的神经元进行免疫细胞化学显示，BAPTA特异性染色与伴随的标记染料在细胞中的分布相似。如对加载了这种指示剂的EDC固定脑片进行共聚焦成像所示，EDC的应用还能有效地原位固定BAPTA的类似物，如钙绿（一种荧光Ca2+指示剂）。综上所述，这些数据表明EDC是一种对多种细胞中的钙螯合剂有效、廉价且通用的固定剂。现在有了合适的固定剂，就有可能在光学显微镜甚至可能在电子显微镜水平上确定此类螯合剂的分布。EDC的两个重要特性源于其对游离羧基的特异性。第一，能够选择性地固定螯合剂而非其AM酯；第二，它作为众多目前可用的其他含羧基螯合剂、染料和pH指示剂的固定剂具有巨大潜力。
