I agree with Jill and Maria - I think also that maybe the tissue is not properly or completely frozen in the embedding medium. I have done cryosections of brain and retina tissue frozen in the OCT and Aquamount embedding media using molds. I was taught to drain all access solution from the fixed tissue by wicking with a tissue or filter paper, then place the tissue into the embedding media in a plastic mold and fast freeze it, then put it in the -80 freezer in a sealed ziplock bag for at least a few hours before cryosectioning it.

To fast freeze, we cooled a small container of isopentane in a bucket of liquid N2, made sure it was sufficiently cold (when it stops vaporizing, takes about a minute), then immersed the mold containing the media and tissue into the isopentane (we used a blunt hemostat clamp to hold the mold during immersion as the isopentane is very cold). The embedding media essentially freezes within a few seconds. I always embedded several tissue samples at a time, so I had a bucket of dry ice nearby in which I put each mold (containing one tissue sample) wrapped in foil and labelled. This kept the embedded samples frozen while I finished the whole batch of samples and then I could put them all in the -80 at the same time (to avoid opening and closing of the -80 over and over - not good for the freezer!). Alternatively, you can also put the mold containing your samples in embedding medium into a bucket of dry ice and let them freeze (I have done this a few times and it worked okay). This takes longer - a few minutes each, but works okay with fixed tissue and you can do multiple samples in parallel.

You have to fast freeze in isopentane cooled in liquid N2 and not directly in liquid N2 is because the emdebbing media and/or the tissue itself can crack when N2 becomes gaseous and bubbles.. 

You should be able to section the tissue immediately after freezing, but the secret that was taught to me was to let it sit in a -80 for some time (even if it is just one hour over lunch) before cryosectioning.

One other issue might be that the tissue is too brittle due to overfixing? You say you store the tissue in 4% pfa after immersion fixation. I would suggest you also try to wash the pfa out and put the tissue in the cryoprotection sucrose solution before storing it in the fridge..

Some more suggestions can be found here:

https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections

and here:

http://www.uab.edu/research/administration/offices/ARP/ComparativePathology/Pathology/Histopathology/TissueSubmission/Pages/Freezing-Tissues-for-Cryosectioning.aspx

Good luck!

I cut mouse brain and spinal cord at -18 and at 0 degree knife with 20 um and 40 um sections.

Dear Maksims,

I have a few questions that may help with troubleshooting your sections.

How did you fix the tissue?  Perfusion or immersion?

How did you freeze the tissue?

Are you able to cut thinner sections (10-12 microns)?

Best,

Jill

I never worked with a cryostat that allow changing the knife temperature. The common situation is to adjust chamber and specimen temperatures. If that is the case, I always use 1 or 2 degrees colder in the chamber than in the sample.

Sometimes changing the knife orientation is also helpful (something between 5º and 10º).

But in any case, I never use embedding medium, it is usually a mess when directly mounting on slide or collecting for free floating.

Hi Stephen,

Thanks for sharing that. Do you mean by 0 degree knife that it is completely vertical?

Dear Jill,

Thanks a lot for your interest :) So:

- the tissue if fixed by immersion and then is stored in PFA in -4 degrees;

- when I start working on the cryostat, I prepare the stage on the sample holder using the embedding medium, i.e. just pour the embedding medium on it, fish out air bubbles if there are any, and then let is solidify properly in the cryostat. After that, I trim the stage to make its surface flat, put the sample on it, and let the sample solidify. Finally, I put the cap from the embedding medium and start cutting when it becomes solid.

- I tried thinner sections some time ago, but that wasn't of much help.

Best,

Max

Hello David, 

I'm working on the CryoStar NX70, and it does allow to control the temperature of both the specimen head and the blade holder. 

As to the use of embedding medium: I'm not sure I can avoid that, as my samples are rather small: approximately 3 by 3 mm large and 300 μm thick. 

Hi Maksims,

I'd second everything which has already been said by others in relation to less cryomatrix being better, also agree with the specimen/chamber temperature gradient (I use -13/-17). One other thing to consider, not all microtome blades are equal; I personally use Feather s35s, other folk use Shandon/Leica blades but I find those to be quite variable in quality, just something else for you to think about. All the best.

Hi Derrick,

Thanks for your suggestions. I'm currently using the ones which my lab got with the cryostat, and I doubt that I'd be able to get any other kind now. Regarding the cryomatrix: I'll try to make it smaller.

Thanks again!

Dear Max,

Thanks for the clarification of your technique.

The reason I asked if you were able to get thinner sections is that I was trying to determine whether your problems were due to poor tissue fixation, poor integration of the sample with the embedding medium, or to the tissue not freezing well.

I think one problem may be that your samples are not freezing evenly, and may even get a bit of freeze-thawing with your freezing technique.  It is difficult to freeze the tissue in the cryostat, since you are just relying on the cold air.  That is why some cryostats come with a special freezing bar or well.

For your embedding method, one thing to try is to place your tissue into a large drop of embedding media at room temperature first in order to replace the sucrose that coats the outside of the tissue, rather than just taking the tissue from the sucrose and placing it on the chuck (sample holder).  Once the sucrose has been diluted with embedding media, place the tissue into fresh embedding media before you place it on the chuck.  This will give you a little more control over how much media surrounds the tissue, and may allow it to freeze more evenly and quickly.  I suggest that you don't add more embedding media on top of the tissue once it has been frozen to the chuck, since that will cause part of the sample to thaw and then re-freeze.

If that doesn't help, then you may want to investigate embedding molds.  I find that since the molds have a flat bottom, it is easy to get a piece of tissue to lie flat while freezing.  Also, you can freeze the tissue in the mold on dry ice or in liquid nitrogen cooled isopentane, so that the tissue plus the embedding medium freezes more evenly.  You can also store the frozen molded tissue in a freezer until it is convenient to section it, rather that keeping the tissue in sucrose for a long time.

Good Luck!

Jill 

Dear Maksims,

I second what was said by Jill - just a few words on my recent experience. It seems that your mail problem is lack of good interface between embedding medium and the tissue. I work on 3 mm biopsies (immersion fixed in 4% paraforaldehyde in PBS, not brain), so similar size to yours. We put the sample on Whartman paper (grade1) to quickly get excess of the 30% sucrose off the tissue, transfer to a few drops of embedding medium (we use Cryomatrix from Thermo Scientific) and then into a cryovial already containing partially frozen embedding medium and immediately cover the sample with 2-3 drops of medium and lower into liquid nitrogen container (that allows for almost immediate freezing. Then we add a few more drops of embedding medium to the cryovial and  store it in a -80degC freezer until cutting. We have no problem with separation of tissue from embedding medium. We use MICROM SEC35p blades and have no problems with sectioning 5-7-15-30 & 50 micron sections. We checked a few different blade types and those seem to be the best for our work. I have listed the source of the embedding medium we use as it seems to adhere to tissue very well - it is not expensive, so it might be a good idea to get one bottle and check if it helps. Our microtome is ancient comparing to yours, but it has a side shelf for leaving tissue on a chuck to adjust to the cutting temperature(at least 1 hour, usually longer). Good luck with your experiments.

yes i meant o degree knife on vertical angle. I use a fairly old leica cryostat. I also let the sample sit in the cryostat for about 15-30 minutes to match the cryostat temp.

I agree with Jill and Maria - I think also that maybe the tissue is not properly or completely frozen in the embedding medium. I have done cryosections of brain and retina tissue frozen in the OCT and Aquamount embedding media using molds. I was taught to drain all access solution from the fixed tissue by wicking with a tissue or filter paper, then place the tissue into the embedding media in a plastic mold and fast freeze it, then put it in the -80 freezer in a sealed ziplock bag for at least a few hours before cryosectioning it.

To fast freeze, we cooled a small container of isopentane in a bucket of liquid N2, made sure it was sufficiently cold (when it stops vaporizing, takes about a minute), then immersed the mold containing the media and tissue into the isopentane (we used a blunt hemostat clamp to hold the mold during immersion as the isopentane is very cold). The embedding media essentially freezes within a few seconds. I always embedded several tissue samples at a time, so I had a bucket of dry ice nearby in which I put each mold (containing one tissue sample) wrapped in foil and labelled. This kept the embedded samples frozen while I finished the whole batch of samples and then I could put them all in the -80 at the same time (to avoid opening and closing of the -80 over and over - not good for the freezer!). Alternatively, you can also put the mold containing your samples in embedding medium into a bucket of dry ice and let them freeze (I have done this a few times and it worked okay). This takes longer - a few minutes each, but works okay with fixed tissue and you can do multiple samples in parallel.

You have to fast freeze in isopentane cooled in liquid N2 and not directly in liquid N2 is because the emdebbing media and/or the tissue itself can crack when N2 becomes gaseous and bubbles.. 

You should be able to section the tissue immediately after freezing, but the secret that was taught to me was to let it sit in a -80 for some time (even if it is just one hour over lunch) before cryosectioning.

One other issue might be that the tissue is too brittle due to overfixing? You say you store the tissue in 4% pfa after immersion fixation. I would suggest you also try to wash the pfa out and put the tissue in the cryoprotection sucrose solution before storing it in the fridge..

Some more suggestions can be found here:

https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections

and here:

http://www.uab.edu/research/administration/offices/ARP/ComparativePathology/Pathology/Histopathology/TissueSubmission/Pages/Freezing-Tissues-for-Cryosectioning.aspx

Good luck!

I agree with Anusha that there is a possibility of the embedding medium or tissue cracking - and for years we were using isopentane. Then we made a few experiments with samples which could be divided to freeze in isopenane and without it - and found out that cooling the tissue by placing it on cooled embeding medium in a cryovial which was dipped into liquid N2, when its surface was still translucent but frozen 2-3 mm below, covering sample with 2-3 drops of medium, waiting to see the medium loose transparency and follow as I have described before - gave us very good results. We had never any problems with tissue or medium cracking, no problems with cutting, and very good tissue morphology in IHC afterwards. Saying that, as  a pathologist, I understand that the source of tissue is very important when choosing the method how to handle it - that is why I have said in my previous post that my samples were not brain but small biopsies from different organs - so they had various amount of connective tissue component which helped protect their integrity. Maksims samples are fragile as they are hippocampus only, not whole brain, so isopentane might be the best option, but I believe that his problem might be mostly due to residual buffer at the tissue/embedding medium interface.

I also agree with Anusha and Maria.  Pick the freezing method that works the best for your tissue.  I have used all three freezing methods with whole brain or eye (with optic nerve attached).  My material is usually perfusion fixed and cryoprotected, and for me and the thickness of the plastic of my molds (available from Polysciences), it seems that powdered dry ice works best.  However, I use a LOT of dry ice--at least two pounds (enough to fill my small styrofoam ice chest).  I keep some of it as a slab, so that it is a big heat sink, and then powder the rest so that it can support the mold.  I wait until the OCT just starts to freeze, and then I carefully start to sift the powder on to the top of the mold, using a plastic teaspoon as a scoop.  Once the top is covered, I quickly heap more powdered dry ice on the mold until it is deeply buried.  I have never had a block crack this way, but I always give it time to equilibrate to the cryostat chamber temperature before sectioning.

For me, it is also a matter of convenience.  At my institution, dry ice is readily available almost 24 hours a day, but having either liquid N2 or isopentane takes planning. In addition I think that I am just used to cutting brain frozen by dry ice, since I have also done a lot of sectioning with a sliding microtome that has a freezing stage.  The freezing stage holds dry ice to keep it cold, and I have cut 15 um to 50 um free-floating sections this way.  In fact, if Maksims tissue was a bit thicker, for example an entire rat hippocampus, I would suggest he try either a vibratome or a sliding microtome, since he needs fairly thick sections and is only interested in Nissl staining.  In theory, he could embed his slices in agar (vibratome) or gelatine/sucrose (sliding microtome) in order to make a thicker block that would work for those machines, but it would also be more difficult to get the face of the tissue parallel to the knife for sectioning.

I think in some ways my method is similar to Maria's in that her cryovials are probably pretty sturdy (made for liquid nitrogen temperatures), and this probably helps buffer the speed with which the liquid N2 cools the sample.  I can also use my thick molds with liquid N2, but it takes a certain technique to dip the mold rapidly in and out of the liquid N2 without submerging it or cracking it.  It also takes longer to reach the correct temperature for sectioning.

So, long story short, use what works best for your samples.

Cheers!

Jil

Dear Jill, This is very true, we have to make do with whatever is available and/or cont effective for our labs, but it leads to good method modifications. I use sledge freezing microtome for thick 50 micron sections for staining as floating sections. Maksims uses a cryostat, so I wasn't suggesting change of equipment. Using sledge microtome has advantage of easier collection of sections and transferring to 24-well plates. Thanks for including the dry ice method - it is a good one for those who have it easily accesible.

I use the same freezing method as Anusha mentioned (dunk isopentane beaker into N2 bath aiming to maintain gentle freezing temperature); find it a pain to do compared to my previous method (dry ice mixed with isopentane) but believe morphology is slightly better. I've known other labs who deal with more CNS samples than us use isohexane as alternative to isopentane but have never tried this personally.

Something I do when I get small samples (which is rare) and has worked well for me, is to break off the lids from 1.5ml/2ml eppendorf tubes, place the sample inside with cryomatrix, top up so it's level, then freeze that. I usually leave them inside the lids till use (so you know which way is up), then pop out prior to mounting/cutting. They seem to mount/cut fine for me, but maybe a 4 sided mold as mentioned previously (as in the ones used for EM?) would be even better.    

1/ How long your samples were in 30% sucrose?

2/ I would not reccomend to keep thel for long time in embedding medie as is take the remaining water away!

3/ How long and in which concentration of PFA you fix them? ( You could try to have 2 % insted of 4% or shorter time in 4%)

4/ Make sections thinner! It's really doesn't metetr 30 micron or 20 micron (for staining ang microscopy) BUT can improve the quality of sections !

5/ Finally - it's looks like your samle is not preventing enoght (with sucrose) for freezing

Dear Stephen, Anusha, Maria, Jill, Derrick, and Natalia,

Thank you ever so much for your advice and sorry for replying only now: it took me some time to test various possible solutions that you suggested, and I did not want to take your time again before some real results would be available. It appears that the problem with the quality of the sections was caused by several factors that had been highlighted in the discussion, namely:

- the poor interface between the sample and the embedding medium,

- the insufficient freezing of the sample, as I used only to freeze it in the embedding medium in the cryostat for an hour or so;

- too high temperature for cryotomy. 

I am very happy that thanks to your suggestions, the quality of the sections has improved greatly. So here's what I changed in my protocol:

- in order to improve the interface between the sample and the embedding medium, I do not transfer the sample directly from the cryoprotective solution onto the sample holder, but briefly dry it on paper (I use Kimtech Precision Wipes for that) so that I would get rid of sucrose crystals on the surface of the sample;

- after embedding the sample in the medium, I put it overnight in the -20 freezer so that it would freeze properly;

- I set the temperature of the chamber at -20 degrees and the temperature of the knife at -30 degrees now.

Many thanks once again! 

Best,

Max

Glad it worked out!

Glad we were of help!

Cheers,

Jill

Glad you got this resolved. All the best.