The overall goal of this procedure is to prepare organotypic transverse slices of the forelimb region of GFP expressing mouse embryos to image spinal nerve outgrowth. This is accomplished by first embedding the embryos in low melting point aros and limiting the area of tissue and aros to the region of interest. The aros block is then mounted for slicing.
Then the embryos embedded in the block are sliced with the vibrator. The final step of the procedure is to remove the aros surrounding the slices and to transfer the slices into culture plates. The slices are then cultured and show outgrowth of peripheral nerves into the periphery, which can be monitored with fluorescence microscopy.
The main advantage of this technique over existing methods like a guillotine or a tissue chopper, is that the slices are generated in a more controlled fashion and show improved viability in culture. Therefore, this method can help in the study of developmental neurobiology, such as looking at the guidance and the outgrowth of spinal nerves, demonstrating the procedure will be Isabel barman. Begin this procedure by preparing 10 centimeter tissue culture plates with six milliliters of culture medium and three centimeter milli cell cm, 0.4 micron culture membrane inserts.
Keep them in the incubator at 37 degrees Celsius and 5%carbon dioxide. Next, heat up 4%low melting point aros in PBS in the microwave, and keep it on a heating plate so that it stays at approximately 37 degrees Celsius. Fill 10 centimeter bacteriological Petri dishes with PBS and place them on ice.
Then set up the Viome cooling device. Store the buffer tray and cooling elements in the freezer until thoroughly frozen to perform embryo. Embedding first dissect embryos from the uterus.
Examine them with an inverted fluorescent microscope and check for GFP expression. Next place embryos on an inverted 10 centimeter Petri dish using watman paper strips to orient them and remove excessive PBS. Apply aros on the embryo in order to fix it.
In the oriented position, wait for the aros to solidify trim excessive aros and if desired tissue surrounding the middle portion of the embryo. Rotate the embedded embryo to the other side. Apply additional aros on the embryonic tissue to ensure that the embryo is completely embedded.
Wait for the aros to solidify. Prepare embryo embedded aros block with clean edges. Finally, glue the sample on a viome chuck with Loctite 4 0 6.
Set up the pre cooled buffer tray and the cooling elements. Place the chuck with the glued tissue on the viome. Add one X-H-B-S-S to the chuck until the tissue is covered.
Insert a pre-cleaned viome blade in the blade holder. Next, prepare transverse slices with a thickness of about 350 to 450 microns. Each slices are cut using an HM six 50 V microtome.
Transfer the slices to tissue culture plates with a shortened glass pasture pipette and keep them on ice. Using a pair of forceps, carefully remove aros from all the slices. Then transfer the slices to the milli cell culture membranes with an arrangement of four slices on each membrane to 10 centimeter culture plates filled with six milliliters of prewarm culture.Medium.
Incubate the slices at 37 degrees Celsius and 5%carbon dioxide until the beginning of time-Lapse imaging and between imaging intervals. Place the tissue culture plate containing Millie cell culture membranes with slices under an upright fluorescent microscope and locate the spinal nerves image spinal nerve outgrowth using four x 10 x or 20 x objectives. Label the orientation of milli cell culture membranes on the microscope stage to reposition correctly during the next imaging time point in case one doesn't have a temperature and carbon dioxide stable.
Environment shown here is an imaging series of spinal nerve outgrowth during 24 hours of culture in a transverse slice of a homozygous tau GFP embryo. The star indicates motor neurons of the ventral spinal cord and the arrow indicates DRG. The upper panel was imaged using a four X objective and the bottom panel with a 10 x objective.
After watching this video, you should have a good understanding of how to prepare organotypic transverse slices of mouse embryos that can be used to image spinal nerve outgrowth.
