Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices.
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.
Kim
Byung G
BG
Department of Neuroscience, NRB Rm EP-04, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007, USA.
Dai
Hai-Ning
HN
McAtee
Marietta
M
Vicini
Stefano
S
Bregman
Barbara S
BS
D000818:Animals; D002232:Carbocyanines; D049229:Dendritic Spines / Q000648:ultrastructure*; D005260:Female; D007834:Lasers; D008853:Microscopy / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D009044:Motor Cortex / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002232:Carbocyanines; C072024:carbocyanine dye DiIC12(3)