The overall goal of this procedure is to investigate proliferation and differentiation of oligodendrocyte lineage cells in an environment that closely resembles what is found in vivo. This is accomplished by first preparing slice cultures of four brain and cerebellum from early postnatal transgenic mice. Single cells in the slices are then imaged over periods of hours to days to visualize the cellular dynamics of their proliferation and differentiation.
After imaging post hoc immunohistochemistry of the cells is used to determine various cellular characteristics. Ultimately, results can show the dynamics of cell proliferation and differentiation under normal conditions and after pharmacological or genetic manipulation. The main advantage of this technique over existing methods like dissociated coal, cultures of neurons and liga incyte lineage cells is that slice cultures allow manipulation of the acellular environment while maintaining tissue cyto architecture.
All animal procedures are following the guidelines and have been approved by the institutional Animal Care and use Committee at the University of Connecticut at least two hours prior to dissection. Prepare tissue culture inserts in six well plates to each. Well add one milliliter of slice culture medium, then transfer the plates to the cell culture incubator at 37 degrees Celsius with 5%carbon dioxide at least 15 minutes prior to the dissection.
Put the dissection buffer on ice and begin bubbling it with 95%oxygen, 5%carbon dioxide gas. Also ensure that all the tools and the tissue chopper are sterilized with 70%ethanol. Transfer the tissue to a sterile 35 millimeter dish containing ice cold oxygenated dissection buffer.
Next, using a razor blade, sever the cerebellum and forebrain follow with amid sagittal cut through the forebrain and cerebellum separating the hemispheres. Now using the manual chopper, make 300 micron thick coronal and sagittal sections of the four brainin and cerebellum. Each animal usually yields six four brainin slices and six cerebellum slices.
Ideally takes slices spanning the anterior one third of the corpus callosum in order to study both gray and white matter regions in the same slice. Transfer the separated slices into freshly bubbled cold dissection buffer on ice in the buffer, using a small dissecting or weighing spatula. Separate the individual sections and transfer them to pre incubated.
Six well dishes containing culturing inserts. Two to three slices can be placed on one culture. Insert pipet off any excess dissection buffer from the surface of the culture.
Insert and transfer the plates to the incubator until they are fixed. The slice medium should be replaced one day after slice preparation, and then every other day until slice fixation as stated in the protocol. Depending on the experiment, use the slices after five to seven days.
In culture. Use only the slices that become transparent after the first few days and discard those that have uneven opaque regions visible to the naked eye. Under face contrast, these opaque regions appear as a clump of dark cells.
If performed correctly, dead opaque regions occur in no more than one to 5%of slices. To perform time-lapse imaging of NG two cell proliferation and differentiation, use three to five day old slices from mice that express EGFP in NG two cells. Do not let the slides get below 37 degrees Celsius for more than 15 minutes.
Per imaging session, keeping the plate closed, use the 10 x objective of an inverted fluorescence microscope to capture images at the first time. Point of the culture. Image four to eight locations on each slice from both gray and white matter areas of the slice.
Also, image useful landmarks like the edges of the slices, particular cells or orientation of white matter tracts for relocation purposes. The previous time point image can be used as a reference image during relocation for NG two cell division and oligodendrocyte differentiation. Capture images every four to six hours for ideally five to seven days.
If the inducible marker NG two cre, ER YFP is used, induce reporter expression using culture medium with 100 nano molar. Four hydroxy tamoxifen dissolved in ethanol reporter activity should appear within one to two days. For fixation, add one milliliter of fixative to the bottom of the culture.
Insert and add another milliliter on top of the slices. Do not squirt the fixative directly on the slices or they may become detached. Fix the tissue for 30 minutes at four degrees Celsius and then using a scalpel.
Cut out the membranes with the slices attached. Using tweezers carefully transfer the slices to individual wells of a 24 well plate loaded with PBS. Next, transfer the slices to a second 24 well plate loaded with blocking solution containing 5%normal goat serum and 0.1%tritton X 100 in PBS.
Allow them to incubate in the blocking solution for an hour at room temperature. Once blocked, move the slices to a plate loaded with primary antibody in PBS with 5%NGS. Allow them to incubate overnight at four degrees Celsius the next day.
Wash the slices in PBS. Then incubate the slices with secondary antibodies in PBS with 5%NGS. Let the incubation go for one hour at room temperature.
Follow this with three more washes in PBS as before and mount the slices with the membrane facing the slide so that it will not be between the objective and the tissue. To analyze the temporal dynamics of oligodendrocyte differentiation, images of NG two cell division were obtained from slice cultures of the forebrain from P eight NG two creeg transgenic mice over multiple days, time-lapse sequences were taken over 122 hours. The phenotype of the divided cells was determined with NG two and CC one antibodies.
Cell proliferation was also assessed using immunohistochemical staining for the cell proliferation marker. KI 67 bait mapping of NG two cells was carried out after induction of CRE recombination in slices from NG two CRE YFP mice. Two days after adding four hydroxy tamoxifen.
YFP reporter expressing NG two positive cells were found in the cultures. Four days later, a proportion of these cells differentiated into CC one positive oligodendrocytes cytes. Mature oligodendrocytes were imaged in cerebellum slice cultures from P-L-P-D-S.
Red mice myelinated Perkin neurons were present in these cultures. Repeated imaging showed the addition of myelinating DS red expressing oligodendrocytes immunohisto chemistry in these slices showed substantial expression of myelin basic protein. Finally, higher temporal resolution imaging revealed stable oligodendrocyte cell bodies with some minor movements in distal processes.
After watching this video, you should have a good understanding of how to prepare slice cultures from both the four brain and cerebellum repeatedly image single cells over days to hours and analyze the fate of image cells using post ho immunohistochemistry. In addition to imaging these slices also allow manipulation of the cellular environment while using fate mapping techniques such as inducible cream recombination in a prepared slice.
