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DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations

Fine neuronal morphology, such as dendritic spines, classically has been studied using the Golgi technique; however, Golgi staining is difficult to combine with other histological techniques. With the increasing popularity of fluorescent imaging, a number of fluorescent dyes have been developed that...

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Autores principales: Staffend, Nancy A., Meisel, Robert L.
Formato: Texto
Lenguaje:English
Publicado: Frontiers Research Foundation 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049322/
https://www.ncbi.nlm.nih.gov/pubmed/21427781
http://dx.doi.org/10.3389/fnana.2011.00014
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author Staffend, Nancy A.
Meisel, Robert L.
author_facet Staffend, Nancy A.
Meisel, Robert L.
author_sort Staffend, Nancy A.
collection PubMed
description Fine neuronal morphology, such as dendritic spines, classically has been studied using the Golgi technique; however, Golgi staining is difficult to combine with other histological techniques. With the increasing popularity of fluorescent imaging, a number of fluorescent dyes have been developed that enable the coupling of multiple fluorescent labels in a single preparation. These fluorescent dyes include the lipophilic dialkylcarbocyanine, DiI; traditionally used for anterograde and retrograde neuronal tracing. More recently, DiI labeling has been used in combination with the Gene Gun for “DiOlistic” labeling of neurons in slice preparations. DiI sequesters itself within and diffuses laterally along the neuronal membrane, however once the cell is permeabilized, the DiI begins to leak from the cell membrane. A DiI derivative, Cell Tracker™ CM-DiI, increases dye stability and labeling half-life in permeabilized tissue, however at much greater expense. Here, the DiI and CM-DiI DiOlistic labeling techniques were tested in side-by-side experiments evaluating dye stability within dendritic architecture in medium spiny neurons of the dorsal stratum in both non-permeabilized and permeabilized tissue sections. In tissue sections that were not permeabilized, spine density in DiI labeled sections was higher than in CM-DiI labeling. In contrast, tissue sections that were permeabilized had higher spine densities in CM-DiI labeled neurons. These results suggest that for experiments involving non-permeabilized tissue, traditional DiI will suffice, however for experiments involving permeabilized tissue CM-DiI provides more consistent data. These experiments provide the first quantitative analyses of the impact of methodological permutations on neuronal labeling with DiI.
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spelling pubmed-30493222011-03-22 DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations Staffend, Nancy A. Meisel, Robert L. Front Neuroanat Neuroscience Fine neuronal morphology, such as dendritic spines, classically has been studied using the Golgi technique; however, Golgi staining is difficult to combine with other histological techniques. With the increasing popularity of fluorescent imaging, a number of fluorescent dyes have been developed that enable the coupling of multiple fluorescent labels in a single preparation. These fluorescent dyes include the lipophilic dialkylcarbocyanine, DiI; traditionally used for anterograde and retrograde neuronal tracing. More recently, DiI labeling has been used in combination with the Gene Gun for “DiOlistic” labeling of neurons in slice preparations. DiI sequesters itself within and diffuses laterally along the neuronal membrane, however once the cell is permeabilized, the DiI begins to leak from the cell membrane. A DiI derivative, Cell Tracker™ CM-DiI, increases dye stability and labeling half-life in permeabilized tissue, however at much greater expense. Here, the DiI and CM-DiI DiOlistic labeling techniques were tested in side-by-side experiments evaluating dye stability within dendritic architecture in medium spiny neurons of the dorsal stratum in both non-permeabilized and permeabilized tissue sections. In tissue sections that were not permeabilized, spine density in DiI labeled sections was higher than in CM-DiI labeling. In contrast, tissue sections that were permeabilized had higher spine densities in CM-DiI labeled neurons. These results suggest that for experiments involving non-permeabilized tissue, traditional DiI will suffice, however for experiments involving permeabilized tissue CM-DiI provides more consistent data. These experiments provide the first quantitative analyses of the impact of methodological permutations on neuronal labeling with DiI. Frontiers Research Foundation 2011-03-01 /pmc/articles/PMC3049322/ /pubmed/21427781 http://dx.doi.org/10.3389/fnana.2011.00014 Text en Copyright © 2011 Staffend and Meisel. http://www.frontiersin.org/licenseagreement This is an open-access article subject to an exclusive license agreement between the authors and Frontiers Media SA, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.
spellingShingle Neuroscience
Staffend, Nancy A.
Meisel, Robert L.
DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations
title DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations
title_full DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations
title_fullStr DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations
title_full_unstemmed DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations
title_short DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations
title_sort diolistic labeling of neurons in tissue slices: a qualitative and quantitative analysis of methodological variations
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049322/
https://www.ncbi.nlm.nih.gov/pubmed/21427781
http://dx.doi.org/10.3389/fnana.2011.00014
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