Optimizing conditions and avoiding pitfalls for prolonged axonal tracing with carbocyanine dyes in fixed rat spinal cords

Chen, Bingkun K.; Miller, Steven M.; Mantilla, Carlos B.; Gross, Lou Ann; Yaszemski, Michael J.; Windebank, Anthony J.

doi:10.1016/j.jneumeth.2005.12.025

Cited by 17 publications

(27 citation statements)

References 27 publications

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“…DiI delivered by gene gun (DiOlistic method) visualized dendritic architecture in the brain slices fixed with 4% PFA (Gan et al, 2000). The speed of diffusion was several fold slower that that of live slices, however, and the duration of fixation needed to be tightly controlled to get satisfactory staining (Grutzendler et al, 2003; but see also Chen et al, 2006). The current study found that lowering the concentration of PFA dramatically increased the speed and extent of DiI diffusion leading to a complete visualization of the dendrites and spinous protrusions.…”

Section: Discussionmentioning

confidence: 99%

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices

Kim

Dai

McAtee

et al. 2007

Journal of Neuroscience Methods

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Visualization of dendritic spines is an important tool for researches on structural synaptic plasticity. Fluorescent labeling of the dendrites and spines followed by confocal microscopy permits imaging a large population of dendritic spines with a higher resolution. We sought to establish an optimal protocol to label neurons in cortical slices with the carbocyanine dye DiI for confocal microscopic imaging of dendritic spines. DiI finely labeled dendrites and spines in slices prefixed (by cardiac perfusion) with 1.5% paraformaldehyde to the similar extent that could be achieved in live preparation. In contrast, fixation with 4% paraformaldehyde severely compromised dye diffusion. Confocal microscopy showed that structural integrity of dendrites and spines was preserved much better in lightly (1.5%) fixed slices than those prepared without fixation. Quantitative measurement revealed that spine density was lower in live slices than that counted in lightly fixed slices, suggesting that fixation is necessary for an adequate evaluation of spine density. The quality of confocal microscopic images obtained from lightly fixed slices allowed us to observe distinctive morphologies such as branched spines and dendritic filopodium, which may be indicative of structural changes at synapses. This method will thus be useful for studying structural synaptic plasticity.

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Section: Discussionmentioning

confidence: 99%

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices

Kim

Dai

McAtee

et al. 2007

Journal of Neuroscience Methods

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“…1,2,3 Chen (2006) suggests that the risk for animal loss associated with an additional surgery, and thereby valuable data, is 10–20 percent. 4 Other drawbacks include the fact that the target pathway is often difficult to reach or inaccessible. 2 Fortunately, within the last few decades, substantial advancements have been established regarding techniques to study neuronal pathways ex vivo by removing organs, or parts of organs, and subsequently applying a tracer.…”

Section: Introductionmentioning

confidence: 99%

“…Upon application to fixed tissues, the dyes possess the ability to diffuse through the fixed plasma membranes of neurons. 4 Axon tracers exhibit the potential to trace neuronal pathways anterograde (away from the cell body), retrograde (towards the cell body), or bidirectionally. 4 Illustrations demonstrating the directionality of axon tracers are shown in figure 1 and 2.…”

Section: Introductionmentioning

confidence: 99%

“…4 Axon tracers exhibit the potential to trace neuronal pathways anterograde (away from the cell body), retrograde (towards the cell body), or bidirectionally. 4 Illustrations demonstrating the directionality of axon tracers are shown in figure 1 and 2. Once in the plasma membrane, carbocyanines have been shown to move bidirectionally and even to adjacent cells through the aqueous medium.…”

Section: Introductionmentioning

confidence: 99%

“…Once in the plasma membrane, carbocyanines have been shown to move bidirectionally and even to adjacent cells through the aqueous medium. 2,3,4,5 Carbocyanine dyes are fluorescent, making visualizing morphology and connectivity along a desired axonal pathway feasible. 4 Figure 3 illustrates the various colors emitted at the corresponding wavelength by different carbocyanines.…”

Section: Introductionmentioning

confidence: 99%

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Histological methods forex vivoaxon tracing: A systematic review

Heilingoetter

Jensen

2016

Neurological Research

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Objectives Axon tracers provide crucial insight into the development, connectivity, and function of neural pathways. A tracer can be characterized as a substance that allows for the visualization of a neuronal pathway. Axon tracers have previously been used exclusively with in vivo studies; however, newer methods of axon tracing can be applied to ex vivo studies. Ex vivo studies involve the examination of cells or tissues retrieved from an organism. These post mortem methods of axon tracing offer several advantages, such as reaching inaccessible tissues and avoiding survival surgeries. Methods In order to evaluate the quality of the ex vivo tracing methods, we performed a systematic review of various experimental and comparison studies to discern the optimal method of axon tracing. Results The most prominent methods for ex vivo tracing involve enzymatic techniques or various dyes. It appears that there are a variety of techniques and conditions that tend to give better fluorescent character, clarity, and distance traveled in the neuronal pathway. We found direct comparison studies that looked at variables such as the type of tracer, time required, effect of temperature, and presence of calcium, however, there are other variables that have not been compared directly. Discussion We conclude there are a variety of promising tracing methods available depending on the experimental goals of the researcher, however, more direct comparison studies are needed to affirm the optimal method.

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2010

Handbook of Biological Dyes and Stains

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Optimizing conditions and avoiding pitfalls for prolonged axonal tracing with carbocyanine dyes in fixed rat spinal cords

Cited by 17 publications

References 27 publications

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices

Histological methods forex vivoaxon tracing: A systematic review

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