Hi Vivica,

Are you sure that process of cryoprotection in sucrose was finished?

I have also other ideas:

- you can use anti-roll plate (glass)

- you can vary temperature in cryostat (if it is possible): chamber temperature should be -15C and specimen head temperature -10C.

Best regards, Anna

Is it possible to seperate the tissue from the "old" Tissue Tec before cutting? I would try to cut the OCT around the tissue and remove it. Then freeze it in the Cryocut again with fresh OCT surrounding the whole tissue.

I saw old, long frozen OCT sometimes behaving like gum. Maybe the connection between OCT and tissue is "broken" by any reason. Freezing is also everytime drying. When the frozen OCT-block is not prevented from drying by any protection it renders to gum-like consistence.

Hi, we usually cut  at 5 microns cerebellar thick slices (220 microns) PAF fixed and cryoprotected. I agree, cryoprotection and cutting temperature are critical...

Try putting the sucrose directly in the fix buffer (20% is OK, time depends on your sample size) and keep it also in the washing step. I currently use OCT and cryostat setted on -20 / -25°C, it  works fine. Samples can be stored at -80°C wrapped in alluminium foil before cutting. Let your samples equilibrate well in the cryostat before cutting. Same conditions for wool rat brains. Good luck!

Vivica, as mentionned below, you have to check:

- the temperature of the cryocut (may be let your sample to equilibrate at the same cryocut temperature by puting it in the cryocut overnight)

- the quality of your tissue teck

- try different positions of anti-roll plate

Be sure that your sample has been in sucrose for 3-4 days before freezing.

Vivica, i sections tissue on a regular basis, try to use the temperature as follows,

inside temp: -17

tissue temp: -19

p.s: use a fine brush to hold the tissue while u section and make sure not to section completely. this will preven rooling up of your tisse. once ur desired section is cut put charged glass slide on the top  to obtain the section.

Hope this helps.

Cheers

Tissue that tends to role up can also be cut like this: Catch the edge with a brush and support it while cutting. Don't cut the block completly, eg stop 2 mm before the upper edge of the tissue. Then turn the wheel backwards. The section will lay onto the block again. On the upper side it is held by the block, on the lower side it is held with the brush. So it is more easy to mount the section onto your slide.

You can watch this method on: http://www.pathologyinnovations.com/index.html

Hi

From my experience this roll happened because the temperature in the cryo machine is too low. So I would start with fixing the temp at the beginning

good luck

Thank you very much for the answers.

I don't think the OCT is the problem, it's quite fresh and good quality. The brush technique does not work for me, the cut gets crumbled any way and separates from the OCT.

But I was told that the tissue did not sink completely in the sucrose solution. What happens if tissue is fixed but not completely cryo-protected and how to solve the problem with this piece? Is there a way to rescue it? What is the maximum time to store in sucrose?

Hello Vivica 

I sent a link with brief method of cryopreservation after the fixation of the sample, .look.

I wish it should be helpful.

You might try adjusting the temperature of your cryostat.  Different types of tissue cut better at slightly different temperatures.  If the tissue curls only after you cut it, you might also look at the temperature in the room that you do the cutting. if the room is too warm, the temperature differential between the air coining in when you slide open the door to attach the tissue and the metal of the cryostat seems to promote curling.

Hi Vivica,

I bilieve that the brain tissue in sucrose has not been well blended with the Tissue Tek (OCT), so you cut well Tissue Tek but the tissue in sucrose  is rolls up. 

Usually, after fixation or perfusio in 4% PFA, do these steps:

•  wash in running tap water for 1 H
•  wash in PBS pH 7.4 for 10 min at 4 ° C
• gently wipe the sample from excessive water
• 30% sucrose for 5 h at 4 ° C
• 60% sucrose for 5 h at 4 ° C
• V / V sucrose 60% + TissueTek (OCT) for 5 h at 4 ° C
• TissueTek (OCT) for 5 h at 4 ° C
• ready for frozen at - 40 ° C

(obviously the times are proportional to the blow up of the sample)

If they are already frozen, this is a tough question.  It depends on how valuable the samples are, but if somebody else sent them to you....I imagine it's hard to simply process more.

What might have happened is that the brains still had a coating of the 40% sucrose when they were frozen.  In the future, I tend to let as much of the sucrose come off as I can, then put the sample into some OCT that I'm not going to use for the freezing (and roll it around gently to coat it).  Then when you move it into your cryomold (filled with fresh OCT) and snap freeze.

Alternatives of how to deal with the current samples:

1.  Start by trying slightly colder temperatures.  Most of my slicing has been done between -14C and -21C, but the temp for a given sample depends on how it was processed, the thickness that you are slicing, and the type of tissue.  Offhand, I would guess that it could help if you went a little colder and/or try slicing a little thicker.  On most cryostats, 12-14um sections would be easier to get than 7um. 

2.  Finally, if you are at wits end and nothing is working, maybe take one of your less important samples and try thawing it, removing it from the current OCT, and moving it to a new OCT cryomold - then refreeze.  This will need to be done gently because tissues become a bit difficult to handle after a freeze/thaw.  But it could be worth seeing first if this helps get better sections, and second, see if this work for the assay that you need.  If you go this route, try it with a single sample from start to finish and if your staining/assay works well, only then should you consider doing it for the rest of the samples.

May be you need use method SealNfreeze with cryo -molds for getting OCT blocks , with than you can freeze your specimens more reliably.

Dmitry Kravtsov

I looked at the SealNFreeze system and have three questions:

What are the inner dimensions of the box, is it made of acetone-proof material if I want to do a colling bath with acetone instead of ethanol and how good are the isolation properties?

I work with exactly the same tissue, although I section it at 16um instead. Don't know if it feels different from 7um. The truth is that I don't like embedding the tissue in tissueTek, so I only using it for mounting the brain to the metal holder. But what you can do is to play around with the temperature. The coldest it is the easier it rolls.

When using OCT-embedded brains, I find that the tissue curls if too cold. Personally I dislike OCT and cryostating. I would recommend M1 from Fisher. I find curling is not a problem. It is a rather different formula.