A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments

M. Tymianski, G. M. Bernstein, K. M. Abdel-Hamid, R. Sattler, A. Velumian, P. L. Carlen, H. Razavi, O. T. Jones

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)175-183
Number of pages9
JournalCell Calcium
Volume21
Issue number3
DOIs
StatePublished - Mar 1997
Externally publishedYes

Fingerprint

Carbodiimides
Fixatives
Calcium
Chelating Agents
Coloring Agents
Neurons
Esters
Ethyldimethylaminopropyl Carbodiimide
Immunohistochemistry
Tissue Fixation
Histological Techniques
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
Spatial Analysis
Acids
Fura-2
Brain
Autoradiography
Anti-Idiotypic Antibodies
Enzyme-Linked Immunosorbent Assay
Electrons

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

Cite this

A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments. / Tymianski, M.; Bernstein, G. M.; Abdel-Hamid, K. M.; Sattler, R.; Velumian, A.; Carlen, P. L.; Razavi, H.; Jones, O. T.

In: Cell Calcium, Vol. 21, No. 3, 03.1997, p. 175-183.

Research output: Contribution to journalArticle

Tymianski, M, Bernstein, GM, Abdel-Hamid, KM, Sattler, R, Velumian, A, Carlen, PL, Razavi, H & Jones, OT 1997, 'A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments', Cell Calcium, vol. 21, no. 3, pp. 175-183. https://doi.org/10.1016/S0143-4160(97)90042-7
Tymianski, M. ; Bernstein, G. M. ; Abdel-Hamid, K. M. ; Sattler, R. ; Velumian, A. ; Carlen, P. L. ; Razavi, H. ; Jones, O. T. / A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments. In: Cell Calcium. 1997 ; Vol. 21, No. 3. pp. 175-183.
@article{2b88197a89304026a5bc2a5474dceb1f,
title = "A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments",
abstract = "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.",
author = "M. Tymianski and Bernstein, {G. M.} and Abdel-Hamid, {K. M.} and R. Sattler and A. Velumian and Carlen, {P. L.} and H. Razavi and Jones, {O. T.}",
year = "1997",
month = "3",
doi = "10.1016/S0143-4160(97)90042-7",
language = "English (US)",
volume = "21",
pages = "175--183",
journal = "Cell Calcium",
issn = "0143-4160",
publisher = "Churchill Livingstone",
number = "3",

}

TY - JOUR

T1 - A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments

AU - Tymianski, M.

AU - Bernstein, G. M.

AU - Abdel-Hamid, K. M.

AU - Sattler, R.

AU - Velumian, A.

AU - Carlen, P. L.

AU - Razavi, H.

AU - Jones, O. T.

PY - 1997/3

Y1 - 1997/3

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0030975427&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030975427&partnerID=8YFLogxK

U2 - 10.1016/S0143-4160(97)90042-7

DO - 10.1016/S0143-4160(97)90042-7

M3 - Article

C2 - 9105727

AN - SCOPUS:0030975427

VL - 21

SP - 175

EP - 183

JO - Cell Calcium

JF - Cell Calcium

SN - 0143-4160

IS - 3

ER -