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fresh-frozen unfixed brain cutting problem

fresh-frozen unfixed brain cutting problem - Histology Forum

fresh-frozen unfixed brain cutting problem - Histology Forum. Talk, discuss and post questions on general histology methods including tissue processing, embedding tissues, sectioning, special staining, enzyme staining and silver staining of samples.


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Old 04-16-2009, 03:22 PM
Pipette Filler
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Default fresh-frozen unfixed brain cutting problem



Hi all,

Currently I am working with mouse brains which are unfixed and fresh-frozen. I am cutting 25um sections. I have a total of 5 brains to do and have already finished 3 without too much trouble. However, the last couple of brains are driving me crazy because they are not cutting very well compared to the other ones. They are very 'crumbly' for want of a better term and always have cracks or generally poor preservation of morphology. I have tried all the standard tricks of different temperatures, section thickness and knife angle to no avail. Any ideas?

Thanks,
Vlad
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Old 11-16-2010, 09:54 AM
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Default Re: fresh-frozen unfixed brain cutting problem

I was wondering why they are crumbly i usually go for 6 to 8 microns sections and problems are mostly because of temparature and angulation of glass slides.Check the temparature properly and try angulating the glass plate again
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Old 02-23-2011, 02:56 PM
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Default Re: fresh-frozen unfixed brain cutting problem

Quote:
Originally Posted by vlad131 View Post
Hi all,

Currently I am working with mouse brains which are unfixed and fresh-frozen. I am cutting 25um sections. I have a total of 5 brains to do and have already finished 3 without too much trouble. However, the last couple of brains are driving me crazy because they are not cutting very well compared to the other ones. They are very 'crumbly' for want of a better term and always have cracks or generally poor preservation of morphology. I have tried all the standard tricks of different temperatures, section thickness and knife angle to no avail. Any ideas?

Thanks,
Vlad
Hey Vlad131,

Brain sectioning can be very finicky and there will always be some sort of freeze artifact. There could be a number of problems and I have a few questions/suggestions.
1) It is generally recommended to fix the tissue first before you freeze them to keep the brain from going to mush.
2) Also, how did you freeze them? The best way to freeze brain sections are isopentane and liquid nitrogen. If you just freeze them in liquid nitrogen or in the OCT compound, it will give the tissue time to create some sort of freeze artifact. The isopentane and liquid nitrogen snap freezes the tissue and doesn't give it enough time to create a freeze artifact. To do this I would fill a Styrofoam cooler with liquid nitrogen and fill a metal container with isopentane and carefully put the metal container with isopentane in the liquid nitrogen. Once the isopentane cools snap freeze your tissue that way. I understand isopentane can be rather pricey and there are other methods that are not hard on the budget.
3) Another option you can get cork and cut it up in small pieces. The cork will absorb some of the water in the brain tissue. Put a small amount of OCT compound on the cork and mount the brain tissue on top. I recommend freeze spray (and it's fairly cheap!) to freeze the tissue to the OCT compound and cork. Embed the tissue with OCT compound and dunk into the isopentane to freeze.
4) If the tissues are shattering or crumbling it sounds like the temperature maybe too low. You said you tried different tricks with temperature, I would recommend a warmer temperature around -10C. One trick you can also use is run your thumb gently across the embedded tissue to warm it up slightly, not too much or it will turn to mush.
5) Another question, this maybe a stupid question but are you using a fresh blade?
6) One last suggestion for the future if you have fresh samples. I'm not sure your source of brain tissue but in mice I have heard of taking the whole mouse head de-calcifying the skull and cutting it on the saggital plane and before embedding and sectioning.

The absolute best reference material I recommend for histology techniques and cryosectioning is: Carson, Freida L. Histotechnology A Self-Instructional Text Third Edition. (ISBN 978-0-89189-581-7). They have an excellent troubleshooting section with each chapter and examples with sectioned tissues.

I hope this helps! Good Luck!

With regards,

Pamela Williams
Laboratory Technician at Protea Biosciences Inc.
proteabio.com
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