[Histonet] Still having Issues with Acetone fixation.

[Lewis, Patrick]

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.

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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?

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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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