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

[Lucas Cahill]

Hi Tim,

Thanks for the insight!
The staining solution is for some experiments on labeling of unfixed tissue
for fluorescence microscopy so I need to immerse the tissue in the solution
prior to formalin. The labels I am using (rhodamine) are more soluble in
alcohol than water so I would like to use them in alcohol.

Lucas

On Mon, Jul 10, 2017 at 11:19 AM, Morken, Timothy <Timothy.Morken at ucsf.edu>
wrote:

> 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?
>
> Best,
>
> Lucas
>
>
> --
> *Lucas Cahill*
> PhD Candidate | Medical Engineering & Medical Physics Harvard-MIT Health
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-- 
*Lucas Cahill*
PhD Candidate | Medical Engineering & Medical Physics
Harvard-MIT Health Sciences & Technology
P: 617-792-0746