The overall goal of this procedure is to perform imaging and analysis of neural progenitor mitosis in live mouse brain slices. This is accomplished by first dissecting brains from embryos. The second step is to prepare brain slices with a vibrato.
Next, the live imaging of brain slices is performed. The final step is to extract information from movies to analyze mitosis parameters of interest. Ultimately, time-lapse microscopy is used to show how progenitor populations of the brain divide in real time.
The main advantage of this technique over existing methods, such as fixed analysis, is that one can gain significant insight into temporal dynamics of neuro progenitor division in live brain slices. It can also be used to understand how genetic perturbations may influence neuro progenitor division. Though we use this method to understand cortical development, the analysis we described can be applied to the study of mitosis of stem cells in other brain regions or even outside the brain.
Demonstrating this procedure will be Louis y, a postdoctoral fellow from my laboratory. Begin this procedure by collecting the mouse embryos and dissect out the embryonic brains, including the hind brains and the four brains with the two cerebral hemispheres. In a bucket of ice, create a hole in the ice for the embedding molds.
Next, pour 3%aros medium into a plastic mold and place that mold into the hole in the ice. Stir the aros medium with the tip of a digital thermometer until the temperature reaches 35 degrees Celsius. Then promptly transfer the brain into the embedding medium to remove excess HBSS at the interface between the brain and the aros.
Carefully and gently rotate the brain repeatedly in the embedding solution. With the forceps, a cushion of gelled aros will form at the bottom of the mold in contact with the ice. Once the cushion can be felt with the tip of the forceps while stirring the brain position the brain with the dorsal side up, the brain should not sink to the very bottom of the mold.
Let the aros harden in the ice for at least five minutes before cutting the corners of the mold with a razor blade. Carefully carve an aros block around the brain and try to minimize the number of cuts to avoid perturbing the embedded brain. With the VT 1000 s Viome generate 200 to 250 micron slices.
Now dilute cyto 11 to a final concentration of 0.5 to one micromolar in the slice culture medium supplemented with growth factors and a 12 well plate incubate slices from one brain in 2.5 milliliters of the staining solution in each well for one hour at 37 degrees Celsius. Then wash the slices in 2.5 milliliters of slice culture medium without staining solution for 20 minutes. To mount the brain sections, place a drop of collagen solution at the bottom of a 35 millimeter glass bottom microwell dish.
Spread it with a pipette tip to match the size of the slice. Then transfer a slice into a drop of collagen. Let the slices incubate at room temperature for 10 minutes before transferring them into a 37 degree Celsius incubator with 5%carbon dioxide.
After 20 minutes at a total of 1.2 milliliters of slice culture medium into the glass bottom micro, well dish 600 microliters of medium at a time and spread it with a pipette tip. Let the slices recover in the incubator for at least one hour and 30 minutes at 37 degrees Celsius with 5%carbon dioxide before transferring them to the incubation chamber of the spinning disc confocal microscope with the same conditions. As for live imaging, use a 60 x silicon oil objective with a working distance of 300 microns and a numerical aperture of 1.3.
For cell imaging image, the cells in a 30 micron Z stack with the center of the Z stack, located about 40 microns below the surface of the slice. If necessary, adjust the laser power and exposure times to limit photobleaching and use the exposure time ranging from 30 to 200 milliseconds depending on the intensity of the cyto 11 signal. Here is representative data that one can achieve from this analysis to identify the mitotic figures in image J.After opening the dataset for one position as a hyper stack, select one Z plane where the tissue and cells look healthy.
Scroll back in time to identify the time point where the cell enters mitosis. The cell may move across Z planes, but the temporal resolution and the Zack parameters described earlier have been optimized to enable this process. In a spreadsheet, record the four D coordinates of the cell in the hyper stack as it enters mitosis.
Knowing these coordinates will prevent counting the same cell several times. Scroll forward in time and record the time when the cell progresses from one phase to another. Document the duration of each mitotic phase for a given cell to conduct 3D reconstruction of the cells and quantitation of rotation during metaphase.
After multiple mitotic cells have been identified, use the coordinates of a cell and scroll forward in time until the beginning. Metaphase rotate the entire hyper stack so that the ventricular border is planer. Use the angle tool to determine the angle to apply for this rotation.
Identify the Z plane where the cell of interest is the most visible in that plane. Use the rectangle selection tool to draw a selection that includes the whole cell. Then identify the Z planes that include the cell of interest and use the image duplicate command to create a sub stack.
In the dialogue box, leave duplicate hyper stack checked the slices. Z parameter corresponds to the Z planes, including the whole cell and the frames T parameter corresponds to the current time point. After that, generate a 3D reconstruction of the cell.
At the current time point. The slice spacing corresponds to the interval between each of the Z plane images in microns. Using the scroll bar, rotate the resulting 3D reconstruction so that the edge of the metaphase plate is visible.
The number of the frame corresponds to the beta angle. Record this number. Then measure the angle between the horizontal and align bys.
Dissecting the metaphase plate perpendicularly. This is the angle alpha record. These angles for all the metaphase time points of the cell of interest.
The angle of rotation between time points t and t minus one equals the absolute value of the difference between an angle at t and this angle at T minus one along the dorsal ventral axis. Mitosis is most easily imaged in dorsal regions of brain slices because the basal process of R GCs is shorter and thus less likely to be severed by a viome. Along the ROS coddle axis slices in the coddle regions of the dorsal cortex give the most consistent results.
These are examples of a good region and a bad region for imaging. In a good region. Nuclei are elliptically shaped and many mitotic cells are observed at the border of the ventricle prior to time-lapse imaging in a bad region, very few mitotic cells are observed at the border of the ventricle and nuclei are round.
Here is an example of a good slice observed with a low magnification microscope prior to live imaging, and here are some examples of cyto 11 labeled cells in different phases of mitosis. Shown here is a montage of a region at the border of the ventricle where two neural progenitors go through the different phases of mitosis marked in different colors Following this procedure. Other methods, like in neutral electroporation of DNA constructs can be performed in order to answer additional questions, including what is the fate of dividing cells and how does the cytoskeleton behave?
It's also best to use secondary methods beyond CY 11 for following mitosis such as H two VGFP transgenic mice. After watching this video, you should have a good understanding of how to manipulate brain slices, what matching parameters are best, and how to analyze the data.
