Ex Vivo Culture and Imaging of Oculomotor Slices from Transgenic Mouse Embryos
Transcrição
Before beginning the procedure, first, confirm the pregnancy by ultrasound on embryonic day 10.5 from a timed mating. Harvest the embryos, spray the abdomen of the pregnant mouse with 70% ethanol, and use scissors to open the abdominal cavity to extract the uterus. Wash the uterus in a Petri dish of ice-cold HBSS before placing the organ in a second Petri dish of fresh ice-cold HBSS.
Under a dissecting microscope, remove the embryos from the uterine horn, and their individual amniotic sacs, placing each embryo on the underside of the lid of a 12-well plate on ice as they are harvested. Use filter paper to remove any liquids surrounding each embryo without touching the embryos themselves, and submerge the embryos in liquid 4% low-melting agarose.
Place the plate lid on ice to solidify the agarose. When the agarose is hardened, flip over the embryos and cover the other side of each sample with additional agarose. When the second volume of agarose has solidified, use a fluorescence dissecting microscope to trim the agarose around each embryo so that each embryo will be oriented properly on the vibratome stage.
The oculomotor nuclei and early axon outgrowths should be fluorescent. Align each embryo so that the nucleus, out-going axons, and eye form a line, and use a razor blade to trim the agarose parallel to this line. Next, fill a vibratome chamber with ice-cold slice buffer, and superglue the first embryo to the vibratome stage so that the blade will be parallel with the oculomotor nucleus and eyes.
When the super glue is dry, submerge the vibratome stage so that the embryo is oriented facing away from the blade, and use a new vibratone blade to obtain 400 to 450-micrometer slices. Use a sterile transfer pipette to transfer each slice into cold slicing buffer as it is acquired, and use the fluorescence-dissecting microscope to select the slice containing the oculomotor nuclei and eyes.
Using a sterile transfer pipette, transfer the slice onto a cell culture, insert in a 6-well plate containing 1.5 milliliters of culture medium per well, and place the plate in a 37 degrees Celsius incubator. Remove the residual agarose from the bottom stage and superglue the next embryo onto the stage, continuing to collect slices until all of the embryos have been sectioned and plated, and image of the slices every 30 minutes by phase contrast and fluorescence microscopy for up to 72 hours.
