[Histonet] Fresh tissue soaked in ethanol solution prior to formalin fixation

Morken, Timothy Timothy.Morken at ucsf.edu
Mon Jul 10 10:19:55 CDT 2017

Luca, sounds like a project to figure that out! 

Anytime you soak the tissue in any percentage alcoholic solution there is going to be some dehydration - substitution of water with alcohol. 

When adding alcohol you will cause precipitation of some of the proteins in the tissue - depends on the alcohol concentration. This does not normally affect immuno or other staining, but can cause some morphological artifacts from the wave effect of the alcohol moving thru the tissue. 

Then if you put back in aqueous formalin, that rehydrate the tissue which may induce other artifacts. A better idea may be to  use alcoholic formalin for the rest of the fixation in the same concentration. However, alcoholic formalin is normally used in a tissue processor after normal fixation with aqueous formalin at which time the tissue should be fixed enough to avoid the morphological changes.  It is an attempt to start the dehydration process earlier in the cycle. 

If the tissue is small, or thin - less than 5mm - it will alcohol fix all the way thru. If larger you could get alcohol fixation artifacts in some areas and not in others where the alcohol does not reach.

What is the staining solution supposed to accomplish as a pre-fixation technique that can't be done later in the process?

An out of the ordinary process like this requires some test studies to see how it affects whatever it is you want to see, in parallel with normal methods.

Tim Morken
Pathology Site Manager, Parnassus 
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center

-----Original Message-----
From: Lucas Cahill via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Monday, July 10, 2017 7:57 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Fresh tissue soaked in ethanol solution prior to formalin fixation

Hi all,

I am using a protocol where fresh breast tissue is immersed in an alcohol based staining solution (50% ethanol, 50% water) for 2-5 minutes before formalin fixation and paraffin processing for H&E, immunohistochemistry, and immunofluorescence. I have not seen differences in subsequent processing on the tissue that I've tested, however, I'm wondering if the 50% ethanol solution on fresh tissue has an effect. I know tissue can be alcohol fixed but this is not standard in clinical breast tissue processing and 2-5 mins does not seem like it would fix the tissue.
Are there any protocols that use an alcohol based solution on fresh tissue prior to formalin fixation that are known not to affect subsequent processing such as immunohistochemistry? Does anyone have any intuition on how this could affect tissue analysis?



*Lucas Cahill*
PhD Candidate | Medical Engineering & Medical Physics Harvard-MIT Health Sciences & Technology _______________________________________________
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