[Histonet] Cryotomy help

[Tony Henwood]

I have never come across this problem but as a suggestion:


  1.
Methanol is often used as an anti-freeze, so I am not surprised that cryotomy is difficult
  2.
Rinse tissues well in a cell culture medium such as RPMI or Hanks to remove as much of the methanol as possible (several changes at least).
  3.
Pat-dry and infuse with Cryo-gel (50/50 with water, then 70%, finally undiluted Cryo-gel). Place on rotor for an hour or so each, until the tissue sinks in the Cryo-gel.

It appears that OCT and sucrose gradients interfere with the Mass Spectrometry due to the high sugar content (see Snijders, M. L., Zajec, M., Walter, L. A., de Louw, R. M., Oomen, M. H., Arshad, S., ... & Clahsen-van Groningen, M. C. (2019). Cryo-Gel embedding compound for renal biopsy biobanking. Scientific reports, 9(1), 15250.)

Lots of luck


Regards,



Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)

Principal Scientist, the Children’s Hospital at Westmead (Retired)

Adjunct Fellow, School of Medicine, University of Western Sydney.

________________________________
From: Colleen Forster via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 13 July 2024 03:19
To: Davoli, Katherine A <katherine.davoli at pitt.edu>
Cc: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Cryotomy help

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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