Visualisation and quantification of dendritic spines in cultured human Medium Spiny Neurons (MSNs)
Quyen Do
Abstract
This protocol describes the visualisation of dendritic spines of human neurons cultured on coverslips in vitro and subsequent quantification using the software Imaris.
Steps
Visualisation of fluorescent dendritic spines in MSNs
Mount a coverslip of Medium Spiny Neurons (MSNs) immunolabelled for DARP32 and neurobiotin onto SlowFade™ Diamond Antifade mountant (follow Protocol: Immunocytochemistry of cultured human Medium Spiny Neurons (MSNs)).
Place slide under under Olympus FluoView FV1000 confocal microscope with argon and solid-state laser with 488 nm and 559 nm excitation, respectively.
Use the 60x oil-immersion objective (NA = 1.40) to image and capture MSNs coexpressing Darpp32 and Neurobiotin as Z-stacks, sampling sequentially at resolution of 1024 * 1024 pixels and at 1.05 µm steps, as optimised by Nyquist sampling theorem.
Use the zoom function (3x) in the Olympus Dendritic FluoView FV1000 software to capture dendritic branches of biotinylated neurons.
Quantification of dendritic spines
Use the 'Surface' module to threshold and segment the confocal images.
Use the 'Filament' module to automatically render the dendrites from images obtained after step 5 .
Choose the spine detection function in the 'Filament' module to detect spines as protrusions from the previously-identified dendritic filament.
Manually exclude putative spines at branch points or disconnected dots.
