The overall goal of the following experiment is to investigate the effect of the apoptotic stimulus fast ligand in the developing cerebellum of wild type and mutant mice. This is accomplished by first dissecting the brain of wild type and knockout mice between postnatal days eight and 12. The second step is to slice the brains in 400 micrometer thick slices and isolate the cerebellum from each slice.
The third step is to culture the cerebellar slices for five days to make sure they are healthy before inducing cell death with fast ligand. The final step of the procedure is to analyze the effect of fast ligand in the different cerebellar cell populations of wild type and knockout mice based on immunofluorescent staining for the apoptotic marker tunnel, combined with cell type specific markers like calbindin for bikini cells. The main advantage of cerebellar life cultures is that it allows the style of cerebellar regional structure and like primary dissociated cultures where the neuronal networks and several organizations are disrupted.
So this matter will allow to answer some key questions in neurosciences, especially because one has the wild type and the mutant mouse. So one can study comparative development, one can study physiology, and also electrophysiology and pharmacology. Though this method can provide insights into the function of fast ligand in cerebellar or cell survival, it can also be used inside of the effects of fast ligand in cortex and hippocampus.
Generally, people new to this method will struggle because in order to obtain healthy cerebellar lysis, it is critical to put them into culture as quickly as possible without compromising their integrity. Therefore, it's important to have a good dissection and slicing skills before attempting the whole process. Where possible.
The steps of the following procedure should be performed in a tissue culture hood prior to dissection. Prepare A CSF media for brain dissection and slice preparation, as well as slice culture media according to the written protocol and store them at four degrees Celsius on the day of dissection. Prepare tissue culture plates for culturing the cerebellar slices.
Add one milliliter of slice culture media to each well of a six well plate. Then use sterile forceps to place the culture plate inserts into each. Well ensure that there are no air bubbles trapped beneath the insert.
Place the culture plate in a tissue culture incubator Set at 37 degrees Celsius and 5%carbon dioxide for at least two hours to precondition the media. Next, bubble the ice cold. A CSF media with carbogen gas saturation of the media with carbogen alters the pH of the solution and produces a color change from red to orange.
Pour a little of the ice cold bubbled A CSF media into a sterile Petri dish and a six well plate. Place these on ice together with the plate of the vibrato that will be used for slicing the brain. The Petri dish containing bubbled ice cold A CSF media will be used for dissecting the brain from a P eight to P 12 mouse pup following dissection.
Use a razor blade to make a sagittal cut in one hemisphere to provide a flat surface for mounting on the vibram plate. The rostral part of the cortex may also be cut. Dry the chilled vibram plate with a paper towel.
Then add a little super glue to the stage and spread it in a zigzag manner with the tip of the super glue tube. To form a thin adhesive layer, use a spatula to transfer the brain to the vibrato stage with the flattened side in contact with the super glue. Once the brain is attached to the vibram plate, add enough A CSF media to cover the brain and add ice below the plate to keep it cold.
Cut the brain into 400 micrometer slices. Then use a plastic pasta pipette with a cut. Widened end to transfer the slices to the six well plate containing A CSF media on ice working under the dissecting microscope.
Use two insulin syringes with small diameter 28 gauge needles to separate the cerebellum from the rest of the brain. If any of the slices have super glue on the edge, use the two syringe needles to remove the glue from the slice. Use the plastic pasta pipette with a cut widened end to transfer the slices onto the top of the filters.
Adding one to three slices per insert ensure that the slices are fully extended and that no liquid is covering them. If necessary, aspirate excess media with the pipette incubated, 37 degrees Celsius and 5%carbon dioxide for 48 hours. After 48 hours, replace half of the media volume with fresh media, making sure that no air bubbles are trapped underneath the insert.
The remaining original medium contains sliced derived trophic factors that are important for cell survival. The apoptotic challenge is performed after five days of culture. Add the fast agonistic antibody JO two to the cell culture media in the wells to be treated.
Leave half of the wells untreated as controls. Once 24 hours have elapsed, aspirate the media from under the wells. Then fix the slices by pipetting one milliliter of ice cold, 4%paraldehyde under the insert and one milliliter of PFA on top of the insert.
Allow the slices to fix in the PFA, the 10 minutes of room temperature after 10 minutes. Aspirate the PFA and wash briefly with two milliliters of cold PBS pipetting. One milliliter on top of the insert and one milliliter below the insert.
Next, aspirate the PBS and pipette one milliliter of 20%ice cold methanol in PBS underneath the insert and one milliliter above the insert. Incubate for five minutes of room temperature, then aspirate the 20%methanol from the well and wash the slice and D well with PBS. As before, pipette one milliliter of 0.5%Triton X 100 in PBS on top of the insert, and another one milliliter underneath the insert.
Incubate for a minimum of 12 hours at four degrees Celsius to perme the slices following permeation. Aspirate the 0.5%Triton X 100 in PBS and pipette one milliliter of 20%BSA in PBS on top of the slice and one milliliter beneath the slice. Block the slices for a minimum of four hours at room temperature or overnight at four degrees Celsius.
When blocking is complete, use clean scissors to carefully cut the piece of membrane insert attached to the cerebellar slice. Then use clean forceps to transfer the piece of membrane into a 24 well plate containing PBS aspirate the PBS and add 250 microliters of tunnel reagent into each well. Making sure that the reagent covers the entire surface of the slice.
Incubate at 37 degrees Celsius for one hour in the dark when the hour has elapsed. Aspirate the tunnel reagent from the wells and replace with 500 microliters of PBS. Protect the plate from light and incubate it room temperature for 10 minutes to wash.
Repeat this process twice after the last wash. Aspirate the PBS and add 250 microliters of a one to 1000 dilution of the calbindin antibody in PBS and incubate overnight at four degrees Celsius in the dark. The next day.
Aspirate the primary antibody solution and wash with PB S3 times for 10 minutes. Then add 250 microliters of secondary antibody diluted one to 500 in PBS. Cover the plate with aluminum foil to protect from light and incubate for at least three hours of room temperature following incubation.
Wash the slices three times for 10 minutes with PBS and using mounting media that contains dappy. Mount the slices onto a glass microscope, slide and cover slip. Image the slices with a confocal microscope using the 488 nanometer and 561 nanometer excitation wavelengths for kale, bendin, and tunnel respectively.
This image shows a healthy cerebellar slice. After two days in vitro, the cell body layer appears translucent and the foliated structure of the cerebellum is clearly seen under the microscope. After seven days in vitro, the foliated structure of the healthy cerebellar slice is still visible.
In addition, dark cell bodies, probably macrophages usually cover the slice. The confocal image seen here is representative of a fast treated cerebellar slice. The bikini cells appear green after staining with a in primary antibody, followed by an Alexa 4 88 conjugated secondary antibody.
The apoptotic cells positive for tunnel staining appear red as the tunnel kit used is labeled with TMR. Red Bikini cells do not appear in a single row, but a clustered forming a like structure While attempting this procedure. It is important to remember to work in tissue culture hood whenever possible, and to minimize the amount of time the cerebral tissue is exposed to possible contaminants like bacteria or fungi.
Using this procedure, other methods can be applied to these slices, including biochemistry and electrophysiological analyses to provide additional information on protein content as well as neuronal excitability. This technique was originally developed to study the effect of fast mediated cell death in Perkin cells carrying mutations that affect the fast pathway. Perial cells rarely survive in primary culture.
So this procedure was the best experimental approach. So I hope after watching this video, you'll have a good idea as to how to make cere barrel slices and how one can study the apathetic stimuli and its effect on different cerebral cell types.
