The overall goal of this procedure is to analyze the development of the burkini cell dendritic tree under various experimental conditions. This is accomplished by first setting up organotypic slice cultures of the postnatal mouse cerebellum. Next, cultivate the slice cultures under various experimental conditions.
For example, test the effect of MGL R one agonists, then fix the cultures and reveal the morphology of individual bikini cells, dendritic trees by anti cal Calbindin immuno staining. The final step is to analyze the Perkin cell dendritic trees. Grown under control or experimental conditions ultimately results from fluorescence microscopy of the immunos stain Perkin cells, and the quantitative evaluation of the size and branching of their dendritic tree show that activation of the neurotransmitter receptor, in particular MGL R one profoundly affects per kinji cell dendritic tree development.
The main advantage of this technique is that almost the complete outgrowth of the Perkin cell end rights occurs during the culture period, and thus, a very complex issue like development of a dendritic tree can be studied in an in vitro setting. This method can help answer key questions in the developmental neurobiology field, such as how activity influences the growth and development of neuronal processes. To begin this procedure, dissect the cerebellum from the brain of a PA mouse pup in ice cold preparation medium under a stereo microscope.
Next, cut the cerebellum into 350 micron thick slices in the sagittal orientation using a McIlwain tissue chopper. After separating the slices, place the slices on millipore cell culture inserts at approximately six slices per insert. Then incubate them in six well plates prefilled with 0.75 milliliters of incubation medium per well in a humidified atmosphere with 5%carbon dioxide at 37 degrees Celsius.
Start the pharmacological treatments at day in vitro three by adding the PMA or DHPG in the desired concentration to the culture medium. Renew the drugs together with the change of the culture medium every second or third day. For culture's fixation.
First, remove the culture medium, then carefully add three milliliters, paraform aldehyde to each well containing the cerebellar slices attached to the membrane of milli cell.Insert. After an overnight cultures fixation, remove the fixative. Subsequently rinse the cultures with phosphate buffer.
Next, add rabbit anti cow binding, diluted in blocking solution to the culture. Dilute rabbit anti calbindin at one to 1000 for selectively visualizing per kinji cells and monoclonal anti new N at one to 500. For selectively visualizing granule cells in blocking solution.
Incubate the cultures with it overnight at four degrees Celsius under slay agitation. On the next day, rinse the cultures three times with 0.1 molar phosphate buffer, dilute the secondary antibodies. Goat anti rabbit, Alexa 5 68 and goat anti mouse Alexa 4 88 at one to 500 in 0.1 molar phosphate buffer, plus 0.1%Triton X 100.
Incubate the cultures with it for two hours at room temperature. After rinsing the cultures three times in phosphate buffer, remove the stain slices from the culture well with a paint brush and mount them on the super frosts plus glass slides. Place the cover slips with an appropriate mounting medium.
Then view the cultures on a regular microscope with epi fluorescence equipment or with a confocal microscope with the fluorescent labeling of bikini cells. The size of the dendritic tree of a given cell can easily be measured if the tree is not overlapping with other cells. Once a bikini cell with a non-overlapping dendritic tree is identified, it is viewed with the 20 x lens.
An image is recorded with a digital camera and then analyzed with an image analysis program. In our lab image Pro plus is used. It allows outlining the dendritic tree of the cell with a single mouse click.
Using the magic wand tool in the measuring mode, the program then computes the area covered by the dendritic tree and exports it to Ms.Xl.Then statistical analysis of the data is done with GraphPad Prism software for the number of branch points. Due to the highly branched and fine morphology of the kinji cells ENDR tree, they need to be counted manually. The 20 x image file of the kinji cells is zoomed in such that it covers most of the screen using Adobe Photoshop.
Then every branch point is counted and marked with a red dot. The identified bikini cells had been photographed repeatedly to monitor the growth of the dendritic tree. From div two to div seven, there was continuous growth in branching of the dendrites.
From div four to div 11, the dendritic tree had expanded substantially.Shown. Here are the examples of the inhibition of the bikini cell dendritic tree development by p KC or mgl R one, stimulation A and D shows the untreated control cells with a well-developed dendritic tree. After nine days of PMA treatment, dendri appear thickened, and the dendritic tree is strongly reduced in size as shown in BNE.
On the other hand. CNF show that after nine days of DHPG treatment, that dendritic tree was greatly reduced in size. Unlike the PMA treatment, many very fine and short side branches were present on the primary and secondary dendrites.
The quantitative measurements here show that both dendritic tree size in the upper bar graph and the number of branch points in the lower bar graph were greatly reduced after PMA or DHPG treatment differences between the controlled dendritic trees and the dendritic trees of bikini cells from treated cultures were significant. Following this procedure, additional methods like electrophysiology can be performed in order to answer additional questions like functional implications of an altered morphology of the dendritic tree. After watching this video, you should have a good understanding of how to set up organotypic cerebellar slice cultures, and how to analyze kinia cells and mythology.
