Bitan:Dendritic spine morphology
Study of dendritic spine morphology
Rat primary hippocampal neurons were used to study the dendritic spine morphology
Reagents and accessories:
DiL stain, streptomycin/penicillin (Invitorgen), paraformaldehyde, Poly D-lysine, cytosine β-D arabinoside (Sigma), neurobasal media, B27 factor (GIBCO), cover slip, capillary glass tube (Fisher Scientific).
Hippocampi were dissected out from E18 pups and cultured for three weeks in neurobasal medium supplemented with B27.
After three weeks, the cells were treated with peptides (3μM) for 3 days.
After treatment the cells were fixed with 4% paraformaldehyde and stained each individual neuron with DiI by microinjection (Eppendorf Cell Technology, Model: 5070) method.
The DiI labeled neurons were imaged at high magnification (100X oil-immersion objective) using the Leica confocal laser-scanning microscope.
Images were magnified further using a 3X zoom so that the morphology of individual spines could be determined and subsequently quantified.
The Z stack images were collected at 0.3 μm intervals to cover the full depth of the dendritic arbors (20-30μm) and then compressed into a single JPEG image. The numbers of dendrtic spines were counted in 100 μm lengths of dendritic branches using Image J software