1. Make sure the vibratome well is full of ice. Take the vibratome chamber out of the ice bucket and wipe the metal surface dry. Place a small dot of tissue adhesive (3M Vetbond #1469) on the lower left-hand corner of the metal square and use the bottle tip to spread the glue out in a fine line. Blow on the glue bottle tip (so it won’t clog) and recap it
2. Pick up one of the hemispheres with a small metal spatula and blot it dry on a piece of filter paper, being careful not to let the brain fall off of the spatula. Being careful not to touch the spatula to the glue, gently slide the brain off the spatula and onto the metal part of vibratome chamber. Position it dorsal side down, making sure it is firmly adhered to the glue
3. Raise the vibratome blade holder all the way up (counterclockwise; 1 turn = 100 microns). Slide the brain chamber into the vibratome. You will need to push it far enough forward so that the blade can clear the front of the chamber. Lock the chamber in place and fill it with chilled CSF.
4. Insert the vibratome blade into its holder. (Hold the metal clip down to open the holder and release it to lock the blade in place.)
5. Use a bent hypodermic needle attached to a small syringe to fish floating glue and other debris out of the chamber
6. Turn on the vibratome and light.Dial the blade down with ten clockwise revolutions.
7. Start the blade moving forward. Hold the switch up to move fast; release it when the blade nears the brain.
8. Use a small paintbrush to guide the brain slice off the blade; don’t let the slice curl up.When the blade clears the brain, turn the vibratome off. Raise the blade with two counterclockwise revolutions. After maneuvering the slice away from the blade, press a bent needle down gently to trim away excess cortex and brainstem. Place the slice to the slide of the holding chamber corresponding to the side of the brain it came from. I generally use a plastic Pasteur pipette to do the transfer.
9. Turn the vibratome on again. It will move backwards, stop it when the razor blade is in front of the brain. Lower the blade with six clockwise revolutions to cut a 400 micron slice (Remember you just went two 100 micron steps up to retract the blade, so you need to go six 100 micron steps down to make a 400 micron slice.). Repeat steps 25-27 until you have sliced all the way through the hippocampus
10. Repeat steps 19-28 with the other half of the brain.(When you get good at making slices, you can slice through both halves of the brain at the same time.
11. While you are waiting, clean and dry your tools and the vibratome well. (A large syringe can be used to suck the water out of the vibratome well.) Put the rat carcass in the freezer and dispose of the vibratome blade in a sharps container.
12. Get your perfusion lines flowing and power up your rig. After you let your slices settle for an hour you’re ready to go!
