[Histonet] Cryotomy help

[Charles Riley]

Thanks for the advice. That's what I feared but was hoping someone may have
found a trick to pull out the methanol to harden the tissue.



On Fri, Jul 12, 2024 at 1:19 PM Colleen Forster <cforster at umn.edu> wrote:

> Once the tissues are in any ethanol or methanol you will never be able to
> get a frozen section. The only way I have been able to salvage anything
> like this was to place the OCT block into 10%BNF on a rotator and let them
> thaw/fix and process into a FFPE block.
>
> If they cannot use FFPE because of the experiment I would say they need to
> start over. This time, freeze the sample, NO METHANOL FIRST. Do the
> fixation as  a post fix OR fix in an aqueous fixative such as 4%para or
> 10%BNF, sucrose protect and then freeze.
>
> Believeme, I have tried to get this to work in so many ways and all
> experts I consulted told me the exact same thing. The mehthaol will not
> allow the sample to freeze hard enough to cut. They don't even want to use
> a slurry with methanol to freeze it the OCT block. The tissue will  absorb
> enough of that methanol to prevent good frozen sectioning.
>
> Good Luck....
>
> Colleen Forster HT(ASCP)QIHC
>
> On Fri, Jul 12, 2024 at 11:09 AM Davoli, Katherine A via Histonet <
> histonet at lists.utsouthwestern.edu> wrote:
>
>> The initial freeze isn't usually the problem.  It's what happens when it
>> warms back up to Cryostat cutting temperatures of -20 or -25.
>> You'd need to get the methanol out of the tissue completely because it
>> causes the support to melt at the temperatures the Cryostat cuts at.
>>
>> And unfortunately I have not found a method that fully removes ethanol or
>> methanol from a tissue sample that would then allow me to freeze and cut
>> it.  If someone else replies with one I will genuinely be delighted because
>> I have this problem a lot.
>>
>> Katherine Davoli, MDiv, HTL(ASCP)cm    (they/them/theirs)
>> Lab Manager, Tissue Culture & Histology Cores, U. Pitt Dept of
>> Ophthalmology
>> 7.373 UPMC Mercy Pavilion        1622 Locust St.,Pittsburgh PA 15219
>> (412) 624-8508   this number cannot receive texts
>>
>> -----Original Message-----
>> From: Charles Riley via Histonet <histonet at lists.utsouthwestern.edu>
>> Sent: Friday, July 12, 2024 12:04 PM
>> To: histonet at lists.utsouthwestern.edu
>> Subject: [Histonet] Cryotomy help
>>
>> I have a researcher who is trying to mass spectrometry on frozen tissue
>> sections. They submitted the sample in methanol and even after sitting in a
>> -80 freezer overnight the samples are too soft to section.
>>
>> Does anyone have any tips on how to harden the tissue for sectioning that
>> won't damage the results for mass spect (I was thinking liquid nitrogen but
>> want to be sure it won't damage the results)?
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>
>
> --
> Colleen Forster HT(ASCP)QIHC
> BLS Histology and IHC Laboratory
> Jackson Hall, Room 2-155
> 321 Church St. SE
> Minneapolis, MN 55455
> 612-626-1930
>
>
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