This study utilizes fluorescence microscopy to visualize the radial migration of cortical neurons in mouse embryonic organotypic brain slices. The methodology focuses on capturing time-lapse images to analyze neuronal migration patterns along radial glial scaffolds.
Take a membrane insert containing transduced mouse embryonic organotypic brain slices, where neurons and radial glial cells express fluorescent proteins, enabling their visualization.
Using fluorescence microscopy, select a slice with fluorescent cortical neurons migrating radially towards the pial surface of the slice using intact radial glial scaffolds.
Transfer the membrane insert with the selected slice into a glass-bottom dish with media to support cellular activity during imaging.
Place the dish into the inverted confocal microscope’s climate-controlled chamber to ensure cell viability.
Adjust the imaging parameters to capture clear images while minimizing photodamage.
Next, configure a Z-stack in the targeted region to visualize cellular structures.
Initiate time-lapse imaging at regular intervals.
Analyze the images to assess the radial migration of the cortical neurons from the subventricular zone toward the cortical plate along radial glial scaffolds, highlighting neuronal migration patterns.
Select a brain slice for imaging by viewing the slices through an inverted microscope. Choose a slice with bright single neurons in the upper SVZ, migrating radially towards the pial surface of the slice.
The presence of fine fluorescent processes of radial glial cells that span the entire cortical plate indicates an intact radial glial scaffold, which is used by migrating cortical neurons.
Transfer the membrane insert with a selected slice into a 50-milimeter diameter glass bottom dish containing 2 milliliters of slice culture medium. Place the dish into the climate chamber of the confocal microscope.
Set the resolution to 512 by 512 pixels. Increase the scan speed from 400 hertz to 700 hertz to increase the frame rate from about 1.4 to about 2.5 frames per second. Use no more than two times averaging.
Define a Z-stack through the electroporated region with a step size of 1.5 microns. Start the time lapse series by taking a Z-stack every 30 minutes. The settings described allow for a sufficient resolution and brightness of the image while keeping photodamage low during acquisition.
繁體中文
