Cryostat Slicing Protocol with Preparation - KravitzLab/KreedLabWiki GitHub Wiki

Material

  • Cryostat (Obviously)
  • OCT Embedding compound
  • A sharp long razor blade
  • An antiroll blade (optional, but recommended)
  • A cryostat chuck
  • Glass pipette or thin brush
  • A 6/12/24 wells plate with 1-2mL of cryoprotectant in each well (FOR FREE FLOATING SLICES)
  • Ethanol absolute (for cleaning)
  • Plastic Molds
  • OCT
  • Brain Sample in PFA
  • Filter Paper
  • 1x PBS
  • Dry Ice
  • Paintbrushes
  • Glass slides with covers
  • Stain (Fluoromount-G™ Mounting Medium, with DAPI)

How to set-up the cryostat

  • Turn on the lights (Lightbulb symbol)
  • Check that the cryostat temperature is at -20 to -22 °c, if not the case, decrease/increase the temperature with the "-" or "+" sign
  • For good measure, press the double arrow symbol to retract the cryostat chuck holder

/!\ IT IS HIGHLY RECOMMANDED TO START WITH THE CRYOSTAT RETRACTED AT ITS MAXIMUM, IT HELPS TO PUT UP THE BLADE AND NOT GET CUT /!\

Not retracted :(

Retracted ! :D

  • Put the sharp blade in the cryostat by first cranking up the lever away from you

  • Take the blade CAREFULLY and wiggle it in the space (green rectangle) with the sharp edge facing the cryostat block

  • Check that your slicing is the adequate size (We usually use 40 µm cuts for brains)

In this picture, the cutting is set at 50 µm. You can increase/decrease it with the "-" and "+" buttons

How to set up the brains

In order to attach the brain to the cryostat block, you will need to first embed the brain in OCT, which is liquid at room temperature but hardens in the cold. One way to do it is :

  • Take the cryostat chunk (green arrow) outside of the cryostat
  • Put some OCT in the middle of the chunk
  • Put the chunk back into the cryostat
  • RAPIDLY place the brain onto the chunk before the OCT solidifies. The brain has to be placed "upward" with the olfactory bulbs pointing up. (NB : You can mount the brain downward with the cerebellum sticking up, it can be more practical when you need to slice most posterior brain regions such as the cerebellum or some posterior nuclei).
  • Wait for around 1-2 minutes that the OCT solidifies (very white color, not shiny) and then add more OCT around the brain to really stick it on the cryostat chunk

There is an alternative way to prepare your brain sample:

  • Grab a mold (small cube) and put the OCT (clear bottle with black cap) on the bottom of the mold. Put a mark on the front of the mold.
  • Take the sample and carefully pour the PFA into the waste container underneath the sink with holes.
  • Grab a filter paper from the regular sink drawer and use it to dry the brain sample.
  • Have the brain right side up and put the anterior portion directly on the side with the mark. Make sure it is leveled.
  • Fill the rest of the mold with OCT to about 2/3 the way up.
  • Place the mold in dry ice and let it sit for about 10 minutes or until opaque.

Once the brain is properly set, you can place it in the chunk holder in the cryostat block

Pull the lever on the left (blue arrow) so you can place the chunk in the holder (Red arrow). Then, with the right lever (Green arrow) you can adjust the anteroposteriority and the medio saggital plane if your brain is not completely straight on the chunk.

Then you can press on the down arrow (or double down arrows) in order to get the cryostat block to get closer to the blade

And with the handle on your right, you can start cutting by advancing the brain every 40 µm closer to the edge. In order to get flattened slides, I recommend keeping the antiroll glass blade over the razor blade. Cutting without the antiroll blade will make the brain slices roll up and make them harder to handle for immunohistochemistry purposes.

After each slice, you can pick up the brain slice with a glass pipette with a melted tip or a thin brush and put the brain slice in the corresponding well.

Once the brain slices are in each well, take them to the bench and prepare glass slides. Grab a larger well and fill with 1x PBS.

Take a pipette, cut the end, and grab all the slices from the respective well from the 12x plate and place them into the larger well. Place the glass slide into the PBS and start working the sections onto the slide faced all in the same direction, using the paintbrush. Label the slide and let it dry completely. Repeat with each sample type.

Add 10 μL of stain onto the slide and put the glass cover on top.

Recurring issues with the cryostat

  • I can't get slices, cutting the brain only makes dust

Solution : The brain is probably too cold. Slightly increase the temperature in the cryostat and wait a few minutes. If it's not enough, take out the brain out of the cryostat for 1 minute and then put it back.

  • I only get chunks of brain slices and not a while slice

Solution : The brain is too hot, decrease the temperature of the cryostat chamber. If it's not working, put the brain back into a box with dry ice for a few seconds.

  • I can get brain slices but they are damaged

Solution 1 : Clean the razor blade, anti roll and metal plate of the cryostat with ethanol absolute, wait a few seconds and then try again.

Solution 2 : Change the razzor blade.

Solution 3 : Remove the brain from the cryostat chuck and embed it again in OCT. If the brain moves while you cut it, you won't get any good slices.

  • I can't get proper slices when I have the antiroll on, but without it the slices look fine.

Solution 1 : Check that the antiroll is properly screwed on and wipe it with ethanol absolute. Solution 2 : Try to go faster with the handle to cut. Solution 3 : Try a different antiroll to see if it works