[Histonet] Still having Issues with Acetone fixation.

[David Kinsley]

Hi Patrick,

I used to use acetone fixation on mouse tissues for cell surface staining.  I used to cut section at 5 to 10 um and let them air dry for 1 hour.  I would then fix in 100% acetone for 20 minutes at 4deg C followed by air drying overnight.  At this point I would either put my sections into wash buffer to begin ihc, or I would wrap them in aluminum foil and store at -80 deg until I was ready to use them.  


I never had an issue with tissue being lost, but I was doing manual ihc.  Are you using an automated platform for your staining?  When I used the Dako autostainer with my acetone fixed sections, the blow step would lift the tissue from the slide, so I had to do manual staining.  The Leica and Ventana platforms are much gentler on the tissues.


If you don't have an ihc instrument, I would suggest using the Shandon sequenza staining system for manual ihc.


Acetone is a much gentler fixative than formalin or alcohol so you need to be very gentle with the tissue.  I found that after I completed my ihc staining if I post fixed the slides in formalin for 10 to 20 minutes prior to counterstaining, the morphology and nuclear detail was greatly improved.


Let me know if this is helpful.


Dave

Sent from Yahoo Mail on Android

From:"Lewis, Patrick" <patrick.lewis at seattlechildrens.org>
Date:Thu, May 7, 2015 at 3:13 PM
Subject:[Histonet] Still having Issues with Acetone fixation.

Hi everyone,

Sorry to keep posting about this,

But I am still having Acetone issues.

I am doing IHC for Cell surface markers that are lost when fixing with etoh or methanol.

When I fix in 100% acetone my epitopes have great signal.

However,  when I fix in 100% acetone, my tissues all damaged by the acetone beyond all recognition.

I can lose up to 90% of my tissue when I fix in 100% acetone for 10 minutes.

But, I get good epitope staining if I have some tissue left on the slide.

When I fix in anything else, I lose 90% or more of my epitope staining, but my tissue morphology looks great.

--
What's the least amount of time I can fix in 100% Acetone for a 5uM section and still have it be fixed?

Is drying after the 100% acetone fixing essential? or Bad for protecting tissue morphology?

--
I am doing IHC on human tonsils cut to 5 uM with a 24 dry after sectioning.

When I fix in 100% acetone, I fix it at 4C for 10 minutes. Then dry for 1 hour in the fume hood

Should it go straight into buffer? Should it be for less time in the acetone?
Should the acetone be Room temp or -20C instead of 4C.

If I was diluting the acetone with buffer, (or etoh) then I could see going straight into buffer afterwards, but because I am using 100% I think that going into liquid right after fixing is too much of a change and my tissues go BOOM.

Please help.

Patrick Lewis


Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115

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