If you want to extract RNA, you might consider using a brain matrix to take a tissue punch from the hypothalamus. You can then put your fresh or snap frozen tissue in an RNA stabilization product like "RNA Later" to preserve the RNA integrity, as it degrades rapidly.

As for cryostat sectioning, putting the brain directly in the -80 freezer is not advised, as you mention- the brain will take a while to reach target temperature, and in that time, cells (and RNA) may begin to degrade, resulting in bad histological quality under the microscope. Additionally, this method likely didn't result in good quality sectioning on the cryostat, as cooling the brain too much results in tissue fragility and "shredded" sections.

Our protocol in the lab for cryosectioning involves placing a beaker of isopentane on dry ice (best to surround the walls of the beaker a bit with ice too), and chill for half an hour. We then cut off the cerebellum, and use cryomatrix or OCT to stick the brain to the cryostat mounting block. We then immerse the mounting block in the chilled isopentane for no longer than 5-10 seconds. You don't want to freeze the tissue for too long, as you run into the same problem as -80 cold tissue- cracking and fragility. Once frozen adequately, place in the cryostat and begin sectioning. If you have the "shredding" issue still, it could be that your tissue is a little too cold, and you can wait a few minutes to let it equilibrate with cryostat temperature. You can also try replace your cryostat blade to try correct this issue, but ensure you use a chilled blade.

Hope that helps! Shota Ikeda

I share in many of the same points recommended above. You need to snap freeze the brain (-80C is too slow). Isopentane on dry ice works well. Wrapping the brain in aluminum and submerge in liquid nitrogen (holding it down with forceps) until you no longer hear it boiling (about 30 s) also works well. Another key point is that you must get it into liquid nitrogen (or isopentane) very quickly once you've decapitated - I have my lab refine their technique to under a minute - to minimize degradation.

From what I'm seeing in the picture, it looks like you may have your cryostat set too cold. We cut fresh-frozen brains at -19C on our cryostat (will differ slightly between systems), and 1C either way will make a large difference.

After perfusion with 4 % paraformaldehyde or 10% formalin solution. I keep the brain in same solution for one day and then shift it to 15%, 25% and 30% of sucrose. For crysection i take

• brain from sucrose solution and cut the brain region rostral/caudal to region of interest (ROI) with a blade.
• I put OCT compound on stage and place brain on it. and then apply OCT on all side of brain and then spray liquid nitrogen. Cover the brain for 10 to 15 min to get the desired temperature. I keep the stage temperature between -18 to -27 then

Important Note: Cutting Temperature should be 1-2ºCbelow when slide start rolling/fell down.

Anna Kalisvaart

Thank you for your detailed reply!

I'll try isopentane with dry ice, and be careful not to immerse too long.

Andrew K Ottens

Thank you for your advice!

I'm very amazed that your lab can isolate brain within a minute.

If your are OK, could you show me the way by video?

I always try isolating brain as soon as possible, but it takes a few minutes...

If I faced shredded pieces when I cut with cryostat, should I raise temperature?

Aamir Sharif

Thank you for your reply.

The way you mentioned is for IHC, isn't it?

I keep your important note in mind. Thanks!

Not having a video handy, I'll try to describe. First, have an aluminum square (10cmX10cm) precut / labeled along with long forceps and a small container of liquid nitrogen. Also have surgical scissors, small scoop/ weighing spoon, pointy forceps and a small rongeur ready. Decapitate by guillotine, cut scalp open along midline (caudal to rostral) and reflect laterally and rostral to show skull. Use rongeur to crush the caudal end of the zygomatic arch, lateral to the parietal bone, on both sides. Then crush the frontal bone forward of bregma (between eyesockets). Then crush the occipital bone, placing rongeur on the lateral aspects and crushing towards midline. Grabbing the occipital bone from the caudal aspect with rongeur, pry skull off. May need pointy forceps to pick away any extra skull pieces. Tease brain out of open skull with scoop/weighing spoon and onto center of aluminum foil square. Fold square without putting pressure to deform brain. Grab end with long forceps, and submerge / hold down into LN2 until boiling stops. With some practice, you can get from decapitation to submerging in LN2 in about 1 min.

From your picture, looking at how the tissue is fracturing and its color, it could be too cold. Since you didn't say what you have the temperature set to, I cannot tell how much you'll need to adjust, but we find -18 to -19C works well with fresh frozen brain. If you happen to be colder, I'd definitely try warming 1C at a time and see if the tissue cracks less. A dull blade can also cause shredding, but your sections don't look "shredded" in that way, but rather they seem just to be fracturing because they are too brittle. However, if the tissue is frozen too slowly and is full of ice crystals (highly likely if frozen by -80C), that too can cause the tissue to become very brittle, like cutting ice shavings. Warming the cryostat in that case will result in the tissue just collapsing on itself since there is no tissue integrity (full of holes from ice damage).

Andrew K Ottens

I appreciate your detailed explanation!

Actually the way is different from ours. I'll try your way of isolating brain.

I'm sorry. Temperature was set at -10℃ in holder and at -12 to -15℃ in blade. As you mentioned, I realized that tissue that are too cold are brittle. Around -10℃ seemed to be suitable. The difference in temperature may be due to difference of cryostat (ours is made by ThermoFisher).

I keep your advice in mind and I'll retry.

Thank you.

It's been a while since I cryosectioned for ISH, but here are a few thoughts...

1) I definitely agree with isopentane on dry ice. Transfer of heat from a solid (the brain) to a liquid (isopentane) is far more efficient than the transfer to a gas (the air in a -80 freezer, or the layer of boiled nitrogen that inevitably forms in a flask of LN when you throw in a piece of tissue at 37degC). IIRC, the brain will turn white just before it reaches the point where it starts to crack--watch for this.

2) If you don't need cell-level anatomical resolution, the suggestion above about punches will simplify your life.

3) Even if you're sectioning for ISH, blocking in a matrix will make it easier--just be careful to have the brain level in the matrix when you slice it. You'd be amazed at how a little bit of angle turns into a change of sectioning plane (and at how much time you can spend staring at an atlas to figure out where your section really is).

4) As another commenter noted, OCT all around will help.

5) Once you've got all that sorted out, if you're still having trouble, it's time to look at the cryostat. You've got a nearly perfect section on the left, and that's encouraging (you probably have a sharp-enough blade, and I don't see any nicks). But the tissue and the cryostat may not be at the same temperature.

Look into: E.Marani. A method for orienting cryostat sections for three dimensional reconstructions. Stain Technology 53: 5, 265-268 , 1978

The article describes your question and more

Lee Koetzner

I'm sorry for my late reply.

Thank you for your detailed comment!

I'll try it!