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Impact of immersion oils and mounting media on the confocal imaging of dendritic spines
- a Graduate Program in Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA
- b Department of Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA
Abstract
Background
Structural plasticity, such as changes in dendritic spine morphology and density, reflect changes in synaptic connectivity and circuitry. Procedural variables used in different methods for labeling dendritic spines have been quantitatively evaluated for their impact on the ability to resolve individual spines in confocal microscopic analyses. In contrast, there have been discussions, though no quantitative analyses, of the potential effects of choosing specific mounting media and immersion oils on dendritic spine resolution.
New method
Here we provide quantitative data measuring the impact of these variables on resolving dendritic spines in 3D confocal analyses. Medium spiny neurons from the rat striatum and nucleus accumbens are used as examples.
Results
Both choice of mounting media and immersion oil affected the visualization of dendritic spines, with choosing the appropriate immersion oil as being more imperative. These biologic data are supported by quantitative measures of the 3D diffraction pattern (i.e. point spread function) of a point source of light under the same mounting medium and immersion oil combinations.
Comparison with existing method
Although not a new method, this manuscript provides quantitative data demonstrating that different mounting media and immersion oils can impact the ability to resolve dendritic spines. These findings highlight the importance of reporting which mounting medium and immersion oil are used in preparations for confocal analyses, especially when comparing published results from different laboratories.
Conclusion
Collectively, these data suggest that choosing the appropriate immersion oil and mounting media is critical for obtaining the best resolution, and consequently more accurate measures of dendritic spine densities.
“… Briefly, number 1.5 coverglass (Zeiss) and slides (Brain Research Laboratories; Newton, MA)
were washed in 70% ethanol and flamed to dry, after which 100 nm red fluorescent microspheres
(FluoSpheres carboxylate-modified microspheres 580/605; Invitrogen, Molecular …”
Copyright © 2015 Elsevier B.V.