Prepare for imaging using a confocal microscope two days after transfection of primary hippocampal neurons. Warm the live cell chamber attachment 60x subjective and lens oil to 37 degrees Celsius and equilibrate the chamber to 5%CO2. Using the eye pieces, identify transfected neurons in the channel containing the cargo protein.
Next, click on scan to image the neurons and identify the axon initial segment of the SCI-5 channel. Set the field of view to a rectangular box of 32 by 128 pixels and move it to image a region of the axon, 50 to 150 microns distal to the axon initial segment. Record the directionality of the cargo transport and the orientation of the ROI, then press save as to save it as file.
Note that the dots on the box will appear at the top and right in subsequent images. Record the side of the image closest to the cell body and the side closest to the axon terminus to ensure correct orientation of the comagraph Polyline. Set the 561 laser power to 1.40, the pinhole to 7.8, the size to 128 pixels square, and the speed to 32 frames per second.
Adjust the gain to visualize the transport of individual cargo vesicles without being obscured by background signal. Select draw rectangular ROI from the ROI dropdown menu and draw the ROI so that it covers the entire field of view. Then right click and select use as stimulation ROI S1.Then in the ND setup tab, click on acquire image to collect the pre stimulation reference image.
Then set stimulation to ROI S1, interval to no delay, duration of 1.64 seconds, and loops to seven. Then click on acquire image to collect post stimulation reference image. Select waiting, no acquisition, two minutes to provide a recovery period for fluorescent cargo to repopulate the bleached ROI.
Select acquisition interval, no delay, duration five minutes, loops, 8, 615. Click the apply stimulation settings button on the ND setup tab, followed by run now. Also collect transport videos from at two neurons for analysis.
Reset the field of view to the entire image and then collect an image of the full neuron in the 561 and 647 channels with the ROI box included. Record the XY coordinates where the image was taken for post fixation confirmation of protein expression. Live cell imaging of a transfected neuron expressing the cargo protein mAPL's Synaptophysin was performed using this protocol.
The external domain of neuro fasin in the Axon initial segment was also labeled. Imaging of cargo transport was performed in the axonal region of interest. Further post imaging immunofluorescence analysis confirmed the co-expression of tau protein and mAPL Synaptophysin.
