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The figure provides the exact protocol to visualize dendritic spines under a confocal microscope. Biocytin was injected inside the neuron using a micropipette, and the slice containing the injected neuron was incubated in 4% paraformaldehyde for 24 hours at 4℃.　The well solution was changed to 50% dilution of the CUBIC-L solution and was incubated for 20 minutes at room temperature. Next, the well solution was changed to the full CUBIC-L solution and was incubated for 40 minutes at room temperature. After incubation, the slice was washed 3 times inside the well using PBS at room temperature. Fluorescent streptavidin with a 1/200 dilution in PBS was incubated after wash for 2 days at 4℃. The slice was, again, washed with PBS 3 times inside the well at room temperature. After the wash, the slice was placed on a slide enclosed with SCALE S4 before observation under the confocal laser scanning microscope.The figure shows the workflow for processing each set of filament data. We defined a spine as any filament over 0.4 µm. A neck was defined as the ratio of the head diameter against the neck diameter exceeding 1.1. A tall aspect ratio was defined as the ratio of the spine length against the head diameter exceeding 2.5. A wide head was defined as the head diameter exceeding 0.5 µm. Lastly, the cutoff between filopodium and thin spines were set at 3 µm.Figure 3　Protocol used to prepare brain slices for confocal microscopy.Figure 4　Algorithm for Spine Classification331