The overall goal of this procedure is to study Neuroblast migration in acute slices of the adult mouse four brain. This is accomplished by first labeling the neuronal precursors in the adult subventricular zone five to eight days after neuroblast labeling, prepare acute slices of the mouse for brain. Next, the time lapse imaging of neuroblast migration is performed.
Ultimately widefield realtime microscopy is used to show the dynamics of neuroblast migration in acute slices. This method can help to answer key question in the field of neuronal development by studying the role of different molecular cues and cellular mechanism involving the cell Migration. Now this method can provide insight into physiological processes regulating neural migration in an adult for brain, but it also can be applied in other systems such as studying neural migration in ischemic brain and during embryonic development.
Begin this procedure by preparing a sucrose based artificial cerebral spinal fluid for cutting the slices and a 32 degree Celsius sodium chloride based A CSF for maintaining the slices until imaging then oxygenate the solutions by continuously bubbling them with 95%oxygen, 5%carbon dioxide. Next, anesthetize the mouse with the fluorescently labeled neuroblast using ketamine xylazine interally. Then quickly prepare the ice cold cutting solution with a jelly-like appearance using liquid nitrogen.
After that, take up 15 to 20 milliliters of ice cold cutting solution with a 20 cc syringe and perfuse the mouse intra Cardi. If the blood vessels need to be labeled instead of perfusing with the cutting solution, inject 200 microliters of dextran Texas red at a concentration of 10 milligrams per milliliter, trans Cardi into the left ventricle of the heart, and wait two to three minutes before decapitating the mouse following trans cardial perfusion, decapitate the mouse rapidly immerse the head in the ice cold cutting solution and move it to the vibrato. Cut the skull with scissors along the sagittal suture from the cerebellum to the olfactory bulb.
Subsequently, gently remove the cranial flaps using a pair of forceps Afterward, excise the coddle part of the brain using a scalpel. Then make two sagittal cuts on the most lateral part of each hemisphere, and cut the brain along the intra hemispheric fissure. Then gently remove the brain using a spatula and place it in ice Cold cutting solution.
Place the two hemispheres separately on a 4%agar block with the dorsal side touching the agar. Next, glue the block with the laterally cut side of the two hemispheres to the platform of a Vibram with the medial side facing up. After that, place the platform in the Vibram chamber.
Fill it with the cutting solution. Then keep the solution oxygenated throughout the slice preparation by bubbling it with 95%oxygen, 5%carbon dioxide. Now prepare the sections of 250 micron thickness with the vibrator.
A low cutting speed and high frequency blade vibration should be used to obtain high quality sections. As soon as a slice is released from the blade, gently remove it from the cutting solution and place it in an incubation chamber filled with oxygenated A CSF maintained at 32 degrees Celsius in a water bath. In this procedure, carefully placed the slice in the imaging chamber of the microscope to avoid drifting of the slice during imaging.
Stabilize it by carefully placing a nylon mesh on top. Make sure that the slice is continuously perfused with A CSF. Next, use a 10 x objective to find a field of interest.
Open metamorph software and ascertain that the nylon mesh does not obstruct the imaging field. Then engage the 40 x objective and adjust the focus. Make sure that there is enough solution between the objective and the slice.
Using the metamorph software set the time-lapse acquisition parameters, such as the duration of excitation, the number of Z planees, the distance between each Z section, the time interval, and the recording duration. Then start the time lapse acquisition. The data is automatically saved by Metamorph as a tiff file With each file corresponding to one time point of the acquired time lapse video to open the movie in Mrs.
Load the acquired images by simply dragging and dropping the first time point image of the video in the program icon. Once the movie is loaded, adjust the brightness and contrast of the signal in the display adjustment window. Set the parameters of the acquired video, such as the voxel size and the time interval in the image properties, and set equidistant time points windows.
After specifying the voxel size, it is possible to see the frame in 3D as well as the time and scale of the video to automatically track the recorded cells. Create a spot for each cell in the field in the surpass window by choosing the spots function, measure the diameter of the cell to be tracked in the slice window. Set the parameters for tracking and proceed.
Inspect the reliability of the detected objects over time. If all the migrating cells are not automatically detected, change the threshold of detection and make sure that all cells have been selected with spots at all time points. The maximum distance and maximum gap size between two spots representing neighboring time points of the same track have to be specified so that the program can connect the time points.
Once the tracks are made, it is possible to filter them and remove the irrelevant tracks by simply deleting them. Tracks can be corrected by connecting and disconnecting different tracks and different time points of the same track As soon as all the corrections have been made. Take a final look at the tracks and export the data, including the cell displacement per time point, track length, displacement length, and track duration to an Excel file.
This video shows the time lapse imaging of the green Neuroblast migration along the dextran, Texas red labeled blood vessels. Note that the migrating neuroblast show saltatory behavior when the migratory phases are interrupted with stationary periods. And this video shows the tracking of neuroblast migration by imas software.
The spheres and lines indicate the cell bodies and migration tracks for individual cells respectively. While attempting this procedure, it's important to remember that good quality acute life slices of adult mouse forebrain should be obtained. In addition, slices should be well stabilized in the imaging chamber by carefully placing a nylon mesh to avoid drifting of the slice during the imaging.
Any changes in the rate of A CSF perfusion, irregular perfusion, or drastic variation in temperature can cause drifting and changes in the focal plane during imaging. After watching this video, you should have a good understanding how to record and analyze neuroblast migration in the acute adult slices. We recommend using short physician interval in the order of 15 to 30 seconds to reliably identify the beginning and the end of the migratory and stationary phases.
