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Author Topic: Frozen tissue in our freezer that my boss wants me to work with  (Read 9628 times)

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Offline funk.106

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Ok, so I have a question that I'm not sure you'll be able to answer because I may not know enough about the problem...but I'm hoping that you can help me with a "well...usually this is how it works..." type of answer.

So I work with human alzheimer's disease brain. Since we are not and MD lab, we basically have to beg for tissue from pathologists and whatnot. At some point in the past (before I joined the lab) we acquired some frozen tissue and it is currently residing in our -80 freezer. All of the tissue that I have worked with up to now has already been embedded in OCT or paraffin; however, this tissue is not. I think the tissue is fixed in formaldehyde. After that, I'm not sure.  I want to make it suitable to cut on a cryostat so I know that I have to put it in the little plastic box and surround it in OCT, but I'm hoping you guys can give me a little more direction so I don't completely screw it up!
Thanks so much!!


Offline Cardio

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #1 on: June 07, 2010, 06:42:00 PM »
I have embedded frozen heart tissue and the only problem I had was sometimes the tissue pulled away from the OCT during sectioning.

I would also make sure that you don't thaw the tissue in a large volume of OCT during embedding phase. I could see more freezing artifacts forming in this case.

Offline funk.106

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #2 on: June 08, 2010, 11:30:54 AM »
So you're saying that when I take it from the -80 and place it in the OCT, I should try to do it quickly enough to not allow it to thaw and to use a small volume so that the OCT will freeze quickly, not allowing the tissue to thaw. Or I should let it thaw, just in a small volume of OCT?

Offline funk.106

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #3 on: June 08, 2010, 02:13:39 PM »
Ok, so I've got another question. I just tried doing this with some control tissue. I decided that you probably meant to keep the tissue frozen. So I filled the cryobox with enough OCT to cover the tissue (kept on wet ice) and then placed the frozen tissue into the OCT and pushed it to the bottom. I found, however that this seemed to create a lot of bubbles, which I imagine is not good for cutting. So I tried to get out as many bubbles as I could and ended up leaving some of the smaller ones in favor of freezing the tissue/OCT. I'm thinking that when I try again, I should maybe do an intermediate OCT step where I place the frozen tissue in some OCT to try to coat it first, then place it in the OCT-filled cryobox. The other thing I was thinking of doing was to just coat the bottom of the cryobox,  place the tissue on top of it and then cover it with OCT by just squirting it directly from the bottle. First, will this help with the bubble issue? Secondly, for future reference, was this the right call to favor freezing over getting out the bubbles or should I be sure to get out ALL bubbles? Third, when I was freezing the tissue/OCT, I used a dry ice/alcohol bath. It seemed kind of slow to me--maybe a minute or two, so not a long time but not as quick as I was expecting. Is there a certain KIND of alcohol that I should be using (methanol vs ethanol, for example)?
« Last Edit: June 08, 2010, 02:27:24 PM by funk.106 »

Offline CanuckPhD

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #4 on: June 10, 2010, 03:52:31 AM »
We cut human brain tissue not embedded in OCT. Just place a dollop of OCT on the chuck, place the tissue on top, use the heat extractor to force the tissue onto the OCT and wait 1 minute. We then just cut the tissue. It takes a little more practise to get good sections but it works fine once you get the hang of it.

On another note. Are you sure all of the proper legal and more importantly ethical requirements have been met concerning this tissue. That is have all subjects or their next of kin approved that the brains can be used for research? This is very important as you can not publish, nor should you use this tissue unless this is the case.

Offline Cardio

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #5 on: June 11, 2010, 09:39:35 AM »
Sorry I have been busy the last couple of days.

First off I use isopentane cooled with liquid nitrogen. Pretty standard protocol for freezing although I wouldn't leave the sample in too long.

You can lock the sample in place with a block of dry ice alone with a small amount of OCT. Once the smaple is locked in place fill the cast with OCT and dip into the cooled isopentane. Example mouse Kidney takes about 10-20 seconds.

IF your confident on the cryostat then you can do it Canucks way.


Offline CanuckPhD

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #6 on: June 14, 2010, 01:18:54 AM »
I just want to clarify that we only cut already frozen tissue this way. For the freezing we use isopentane cooled in a dry ice/ethanol bath. Freezing fresh tissue in the cryostat should never be done as you will have serious freezing artefact.


Offline funk.106

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #7 on: June 24, 2010, 10:28:36 AM »
I have embedded frozen heart tissue and the only problem I had was sometimes the tissue pulled away from the OCT during sectioning.

Ok, so I tried sectioning the tissue that I embedded in OCT and I found exactly this. It was as if the OCT wasn't sticking to the tissue. I had practiced on some control tissue for just such an issue, but have you found anything that helps prevent this? Also, is there any way to save the tissue that is already embedded in OCT for in case I have this same issue on more valuable tissue?

Offline CanuckPhD

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Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #8 on: June 24, 2010, 11:05:48 AM »
In my experience once the tissue is frozen it is difficult to put in OCT. The OCT doesn't bind to the tissue and just makes cutting difficult. Just try cutting the frozen tissue after adhering it to the chuck with a dollop of OCT. This usually works for me. In fact when we do some analysis the OCT interferes with the MS so we only cut the tissue in this manner.

So we take the block of frozen human brain, place a good sized drop of OCT on the chuck, quickly place the frozen tissue on it, put the heat extractor on top and wait 2-3 minutes. Then just cut it (we usually do 10um thick sections). The morphology is decent and once you get used to it the cutting is fine.

Good luck.

Re: Frozen tissue in our freezer that my boss wants me to work with
« Reply #8 on: June 24, 2010, 11:05:48 AM »