A Modified Roller Tube Method for the Long-Term Culture of Rodent Brain Slices
Transcrição
To begin, prepare the roller tube rack, and make a jig to assist with drilling a hole in the roller tubes, as described in the accompanying text protocol. Using the jig, hold in position a flat-sided 1-centimeter plastic culture tube so that the flat side is facing up. Then, drill a 6-millimeter diameter hole with the center 1 centimeter from the bottom and centered between the sides of the tube.
With the swiveling deburring tool, smooth the edges of the hole and make four grooves on the inside edge of the hole to facilitate draining of the hole during rotation. Next, rinse the tubes with 70% ethanol, and then air-dry them in a biological safety cabinet. Sterilize the tubes in some 12-millimeter punched adhesive silicone rubber disks for 40 minutes under the UV lamp.
After 20 minutes, reposition the tubes and disks so that all of the exposed surfaces are sterilized. Next, peel off the white backing from the adhesive disk. Align the holes and affix the silicone rubber to the outside of the tube.
Once the slices have been obtained, place 2 microliters of chicken plasma on the center of the photo-etched side of a prepared coverslip. Spread the plasma slightly to achieve a 3 to 4-millimeter diameter spot. Then, use a sterile, narrow-tip spatula to lift a prepared brain slice. Use closed forceps to keep the slice on the spatula tip while lifting the slice. Touch the spatula to the plasma spot on the coverslip and, with closed forceps, push the slice onto the coverslip.
Add a thin layer of chick plasma on the coverslip before placing the slice and a minimal amount of thrombin-treated plasma to cover the slice. If the slice floats, it will not remain adhered when the clot is removed.
Next, mix 2.5 microliters of plasma and 2.5 microliters of thrombin in a separate tube. Quickly place 2.5 microliters of this mixture over and around the slice, and pipette up and down gently to mix it. Remove the clear plastic covering from the exposed side of the silicone rubber adhesive previously affixed to the roller tube. Then, place the coverslip with the brain slice onto the adhesive, aligning the slice within the hole.
To ensure adhesion, apply soft even pressure to the coverslip with the thumb by pressing the coverslip down evenly and holding it for about 1 minute while transferring it to the biological safety cabinet.
The pressure on the coverslip to adhere it to the tube without leaking must be just right and maintained for long enough to eliminate air channels in the sealant. Too much pressure will crack the coverslip.
In a biological safety cabinet, add 0.8 milliliters of complete neurobasal A culture medium to each tube. Then, flow a 5% carbon dioxide, 95% air mixture through a sterile cotton-plugged pasture pipette held securely by a clamp. Use this to flush the roller tube with the gas mixture and rapidly cap the tube as it is withdrawn from around the pipette.
Next, label the tubes with the slice number and rack number. Insert the tubes into a roller rack, ensuring that they are geometrically balanced. If there is an odd number of tubes, add blank tubes to balance.
Place the racks in a 35 degrees Celsius roller incubator with rollers turning the roller rack to keep the medium in the bottom of the tubes. Tilt the incubator back approximately 5 degrees by lifting its front onto a board.
