[Histonet] Still having Issues with Acetone fixation.

[Julio Benavides]

HI David,

how did you do the post IHC fixation? immersion in buffered formalin 4%?

Looks interesting

Thanks a lot

Julio



David Kinsley <davidkins at yahoo.com> escribió:

> 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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