Kindler, Stefan and Kennedy, Mary B. (1996) Visualization of autophosphorylation of ^(Ca^(2+))/calmodulin ^(-dependent) protein kinase II in hippocampal slices. Journal of Neuroscience Methods, 68 (1). pp. 61-70. ISSN 0165-0270. http://resolver.caltech.edu/CaltechAUTHORS:20120424-154223612
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We describe a method for visualizing the relative spatial distribution of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) in neuronal subcompartments within hippocampal slices. The method employs a monoclonal antibody recognizing only autophosphorylated CaMKII, and an affinity-purified polyclonal rabbit antisera recognizing only nonphosphorylated CaMK II (Patton et al. (1993) Mol. Biol. Cell, 4: 159-172). 50 microns sections cut from fixed 500 microns hippocampal slices are double-labeled with these antibodies bound by secondary antibodies coupled to fluorescein and Cy3, respectively. The distribution of the two antigens in identical optical sections is recorded by dual channel confocal laser scanning microscopy (CLSM). The digital images are analyzed with the program MacPhase to determine the relative levels of staining with antibodies to phosphokinase and antibodies to nonphosphokinase in subcellular domains of neurons. Comparison of data from paired control and experimental slices reveals the spatial distributions of changes in levels of autophosphorylated CaMKII produced by pharmacological treatments. We are able to detect and spatially resolve differences in levels of autophosphorylation of CaMK II between slices subjected to Ca2+ depletion (low autophosphorylation) and slices treated with a phosphatase inhibitor (high autophosphorylation).
|Alternate Title:||Visualization of autophosphorylation of Ca2+/calmodulin -dependent protein kinase II in hippocampal slices|
|Additional Information:||Copyright © 1996 Published by Elsevier B.V. Received 20 October 1995. Revised 23 February 1996. Accepted 30 April 1996. Available online 18 May 2004. We thank L. Schenker and M. Kiely for technical assistance and S. Fraser for valuable advice. This work was supported by an International Human Frontier Science Program fellowship to S.K. and US Public Health Service grants MH49176 and NS17660 to M.B.K.|
|Subject Keywords:||Synaptic plasticity; Protein phosphorylation; Immunocytochemistry; Long-term potentiation; Confocal laser scanning microscopy; Immunocytochemistry; Synapse; Synaptic transmission|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Melanie Stefan|
|Deposited On:||25 Apr 2012 16:52|
|Last Modified:||09 Apr 2015 23:56|
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