Preparation of Organotypic Slice Cultures from a Rat Pup's Hippocampus
筆記録
Using a clean scalpel blade, cut the two hemispheres of an isolated rat brain apart. Transfer the left hemisphere back to the large Petri dish, and place the other, pia side down, in ice-cold dissecting solution.
View the medial face of the right hemisphere and identify the edge fornix, a prominent band of white matter along the medial edge of the hippocampus. Once identified, cut through the fornix using only about 0.5 centimeters of the scalpel blade. Using two micro-spatulas, remove the hippocampus by placing the right hand spatula on the brain stem and lifting the overlying cortex with a left hand spatula.
Gently lift the cortex to reveal the lateral ventricle and medial surface of the hippocampus. A white curved line, the fimbria, should now be visible. Align the curvature of the spatula with the curvature of the fimbria, and gently press the spatula under the fimbria. Slide the spatula left and right along the rostral caudal axis before lifting in the dorsal direction to remove the hippocampus.
Transfer the hippocampus to a separate small Petri dish with ice-cold dissecting solution. Repeat this procedure on the left hemisphere to remove the left hippocampus. Once isolated, carefully transfer the hippocampi to the tissue chopper stage. Arrange them adjacent and parallel to each other and perpendicular to the axis of the chopper blade. Use a paintbrush to position the tissue and add a few drops of the dissecting solution on top of the hippocampi.
Cut the tissue in 400-micrometer slices without pausing to remove individual slices. After the whole hippocampus has been cut, use a paintbrush to gently transfer the sections to a small Petri dish with dissection solution. Under a dissecting microscope, carefully separate the floating slices using a micro-spatula and paintbrush.
Next, remove a culture plate previously prepared with a culture insert from the incubator and place it into the laminar flow hood. Using a fire-polished Pasteur pipette, draw up four to five slices and place them onto the apical surface of the culture insert membrane. Use a paintbrush to adjust their spacing, leaving space between individual sections and the border of the culture insert.
Once in position, remove the excess dissecting solution from the apical surface of the membrane. Place the culture plate with serum containing culture medium and the hippocampal slices back into the incubator at 35 degrees Celsius and 5% carbon dioxide.
Feed the cultures in a sterile laminar flow hood two days post-dissection. First, aspirate out the old culture medium and then add 1 milliliter of fresh serum containing medium to the wells using a sterilized glass pipette. Gently replace the culture insert, and take care to remove any air bubbles that may have formed underneath the membrane surface. After the first feeding, change the medium every other day.
