Freezing Microtome
- Microtome
- Dry Ice
- Paper towel
- Brain Slice
- Razor Blade
- Paint brush
- 12 Well plate
- Larger well plate
- PBS (Phosphate-Buffered Saline)
- Mounting Medium (OCT)
- Aluminum Foil
- Styrofoam box
- Put dry ice around the center block platform to chill it. While this is happening, remove the brain from the 30% sucrose solution. Dry the brain with a paper towel.
- Use a razor blade to block off the cerebellum of the brain.
- Add a small blob of OCT onto the platform
- Before the blob of OCT completely freezes, put the brain cerebellum side down on the OCT. Try to level the brain completely. You can use extra OCT the base of the brain to secure it.
- Wait until the brain freezes completely (it will turn pale white). At this time, grab a 12-well plate and a larger well plate filled with 1x PBS.
- You can adjust the thickness of your slices by changing the knob labeled "Z" below. Crank the knob labeled "Y" until the top of the brain just touches the blade. It is now ready for slicing.
- Using the paintbrush, coat the blade with some PBS to ensure it will cut smoothly. Pull the handle back and forth to create each slice. Each pull will advance the stage upwards automatically. Make sure to pull the blade completely back to ensure advancement of the stage. You may want to discard the first 20-30 slices (usually, we aren't looking at olfactory areas) until slices are large enough to collect.
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Collect your slices with a paintbrush dipped in PBS:
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Sections will flatten out when you float them in PBS. Collect sections in a 6 or 12 plate.
- Once done, remove the brain from the mounting media with the razor blade. Clean area with ethanol.