Preparing Free-Floating Slice Cultures from an Adult Human Brain
Transcripción
Transfer the specimen to a Petri dish containing slicing solution. Using fine surgical tools, carefully remove as much of the remaining meninges as possible. Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design, and use a number 24 scalpel blade to trim a flat surface to be the base that will be glued to the specimen disk.
Using a disposable plastic spoon and delicate paint brushes, collect the fragment from the Petri dish, and dry off excess solution with filter paper. Next, use super glue to attach the tissue to the vibratome specimen dish until it is firmly adhered to the dish and in contact with the agarose block.
Place the vibratome specimen disk into the vibratome buffer tray. Lock the knife holder in place with the razor blade firmly fixed. Add slicing solution and ensure that it is covering both the specimen and the blade. Then, begin slicing the specimen into 200-micrometer slices.
Transfer the slices from the buffer tray to a Petri dish containing slicing solution and trim any loose edges and excess white matter to a proportion of approximately 70% cortex and 30% white matter. In a laminar flow cabinet, add 600 microliters of culture medium to each well of a 24-well plate and incubate at 36 degrees Celsius with 5% carbon dioxide for at least 20 minutes prior to plating the slices. After this, use a paintbrush to plate one slice in each well.
If there are any unused wells in the plate, fill them with 400 microliters of sterile water. Incubate at 36 degrees Celsius with 5% carbon dioxide. Supplement 10 milliliters of the previously prepared culture medium with brain-derived neurotrophic factor at a concentration of 50 nanograms per milliliter.
After 8 to 16 hours, remove 333 microliters of the conditioned medium from each well and add 133 microliters of fresh BDNF-supplemented medium. Replace one-third of the conditioned medium with fresh BDNF-supplemented medium every 24 hours.
