Emccd andor du 897 camera

Hippocampal cultures were plated in glass-bottom dishes (MatTek), transduced with virus expressing shRNA, or treated with drugs, transfected with EB3-tomato reporter at 7 or 8 DIV and imaged 24 h later, or with mEOS3.2 constructs at 5 DIV and imaged 3 days later. For analysis after expression of CDK5RAP2 fragment 51–100, cultures were co-transfected with plasmid encoding EGFP, or EGFP-CDK5RAP2 fragment 51–100 together with EB3-tomato reporter (2:1 ratio) at 6 DIV and imaged 48 h later. Live imaging of EB3 comets or mEOS3.2 alone or fused to Eg5 or α-tubulin was performed in the dendrites of random transfected cells, using an Olympus IX81 microscope equipped with Yokogawa CSU-X1 spinning disc and a temperature-controlled CO₂ incubation chamber. Image stacks were acquired with 100×/1.4 OIL immersion objective and an iXon EMCCD Andor DU-897 camera, using iQ2 software. Fluorescent images with pixel size of 0.14 μm were taken at 1 s intervals during 2.5 min for EB3 comets in dendrites. Fluorescent images with pixel size of 0.14 μm were taken at 10 s intervals during 4 min for mEOS experiments. mEOS3.2 was photoconverted using a 405 nm laser at 5% for 1 s.

Hippocampal cultures were plated in 0.1 mg/ml poly-D-lysine coated glass-bottom dishes (MatTek), transduced with virus expressing shRNA at 1 DIV and transfected with EB3–Tomato reporter-expressing plasmid at 3DIV. EB3-comets of randomly transfected neurons were imaged 24 h later using an Olympus IX81 microscope equipped with Yokogawa CSU-X1 spinning disc and a temperature-controlled CO₂ incubation chamber. The proximal dendrite region was defined as ∼20 µm from the soma and distal dendritic region was defined as ∼20 µm away from the dendritic tip. Image stacks were acquired with 100×/1.4 oil immersion objective and an iXon EMCCD Andor DU-897 camera, using iQ2 software. Fluorescence images with a pixel size of 0.14 μm were taken at intervals of 1 s for 150 s. Multiple planes were imaged with a step size of 0.2 μm. Z-stacks were acquired by using ImageJ software (NIH; https://imagej.net/ij/).