This article describes a method for transfecting neurons with plasmids encoding vesicle-specific fluorescent proteins. The procedure allows for the visualization of vesicle movement within neurons using fluorescence microscopy.
Take the desired plasmid in serum-free media. This plasmid encodes vesicle-specific proteins with fluorescent tags.
In another tube, take the transfection reagent diluted in serum-free media.
Mix the two solutions and incubate.
The transfection reagent combines with the plasmid, forming a complex.
Take a well containing adherent neurons in the media with serum. Add the complexes and incubate. The complex facilitates the plasmid's entry into the cell.
Discard the media to remove non-internalized complexes.
Add fresh media and then incubate.
The plasmid enters the nucleus to be transcribed and is then translated into vesicle-specific fluorescent proteins that label the vesicles in the neurons.
Using a fluorescence microscope under controlled temperature, acquire images at specified time intervals.
To analyze the images, define tracking parameters.
Identify the vesicle of interest and record its coordinates in each of the images.
Compile the data to visualize the vesicle's movement.
In this procedure, mix 4 micrograms of plasmid with 200 microliters of serum-free medium. In a separate tube, dilute eight microliters of the transfection reagent in 200 microliters of serum-free medium. Then, incubate the solution for 5 minutes at room temperature. After five minutes, mix the plasmid solution and the transfection reagent solution, and incubate the mixture for 20 minutes at room temperature. Following that, add the mixture to the coated glass bottom dishes.
Incubate the neurons at 37 degrees Celsius for 30 minutes. Afterward, replace the medium with 2 milliliters of cerebral cortical culture medium, and incubate the neurons at 37 degrees Celsius for one to two days.
For imaging, use a fluorescence microscope equipped with a CCD camera with the 40x objective lenses. Keep the temperature at 37 degrees Celsius using an incubation system. Acquire neuron images at 1 frame every 5 seconds over a 100-second period controlled by the image acquisition software.
To analyze the images, open all the sequential images in ImageJ software with manual tracking. To combine the sequential images, choose Image, then Stacks followed by Images to Stack, and then click OK. After that, choose Plugins, then Manual Tracking. A tracking window will pop up. Click on the checkbox of Show Parameters, and define the tracking parameters in the parameters section.
Then, click Add Track to start a new track. After determining the vesicles of interest, click on the center of the signals in the sequential images to record the XY coordinates. Repeat this procedure to collect the XY coordinates of the vesicles from all sequential images. Each image automatically advances to the successive image after the reference point is selected.
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