[Histonet] Fixation time and ISH

[Rosa, Taylor]

Hi Greg,

For any that have ISH staining, we add to the protocol that fixation should be 24-32h. Tissues are transferred to 70% EtOH after that. One thing to note is that for brain tissue, it should not be in 70% for longer than 24 hours, because tissue artifacts (vacuolization) will occur.

Thanks,

Taylor Rosa

-----Original Message-----
From: Greg Dobbin <greg.dobbin at gmail.com> 
Sent: 07-Mar-2022 11:17 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Fixation time and ISH

Background:
I read on a vendor website that tissues stained using In situ hybridization should not exceed 32 hours fixation. Greater than 32 hours can produce false negative results because the validated retrieving protocol can be inadequate.

We in fact have seen evidence of this in our lab (mystifying negative kappa/lambda staining) and we are beginning to realize that over-fixation May we’ll be the issue for us (eg weekend processing).

I am proposing that we do what was common practice many years ago. That is, transfer the properly fixed and decalcified bone marrow cores into 70% ETOH until they can be processed into paraffin.

My questions:
Is anyone else doing this??

Is 70% ETOH still a viable option for labs in this situation?? And…

Is there another idea and/or more information out there that could help us in this regard??

Thank you,
Greg
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*Greg Dobbin*
1205 Pleasant Grove Rd
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