[Histonet] Acetone fixation problems with OCT Tissues

[Julio Benavides]

  To dry them overnight before fixation is not a bit too risky? I  mean,
wouldn´t the tissue degradate? I normaly fixed the section as soon as I
get it into the slide.

Just a thought. I am no expert in cryostate, just an occasional user.

Julio

"Lewis, Patrick" <patrick.lewis <@t> seattlechildrens.org> escribió:

> Hi Everyone,
>
> I am still having issues with my IHCs with Acetone fixation.
>
> If I fix in 100% Acetone, I get IHC staining, but my tissues are 50-90%
> destroyed.
>
> If I fix in 4% paraformaldehyde, or 10% NBF or (95%  Etoh and/or
> Methanol with Acetone) I lose the epitopes I either get no staining or
> very  weak staining, but the tissue morphology look fine.
>
> I just tried an acetone gradient where I cut the tissues at 5 uM and
> dried them overnight, then fixed for 10 minutes in 100% acetone, then
> fixed in 95% acetone for 1 minute, then fixed in 70% acetone for 30
> seconds, then quick rinsed in H20, then washed as normal in DPBS pH 7.4.
>
> I did 4 slides, 2 slides with one company's Charged slides ,and 2 slides
> with another company's charged slides.
>
> One company's slides look completely destroyed, the others may turn out,
> it was hard to tell how much damage there was.  I'll know tomorrow when
> I finish staining and Hemotoxylin them.
>
> Patrick Lewis
> Research Associate II Bench
> Seattle Childrens Research Institute
> 206-884-1115
>
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