FW: [Histonet] fixing immuno and antibody tissues

From:Patti Loykasek


------ Forwarded Message
From: Patti Loykasek 
Date: Tue, 10 Aug 2004 15:06:23 -0700
To: George Cole 
Subject: Re: [Histonet] fixing immuno and antibody tissues

What a wonderful viewpoint on this conundrum. I don't pretend to have all
the answers, but here's what I think I know! The pathologists prefer to make
diagnoses on formalin fixed tissues - it's the "gold standard" for
morphology. The pathologists are familiar with the artifacts caused by
routine  formalin & processing. In many instances these days they are making
diagnoses on needle biopsies, and there is simply not enough tissue to
freeze & submit for routine processing. It is in this milieu that recovering
antigens became important. First with enzymes, and more recently with heat
pretreatment employing various buffers. Definitely have to be careful that
you are getting actual staining & not some type of artifact caused by the
heat pretreatment. Sometimes, I think we are too quick to pretreat. Itıs
always good to try to get an antibody signal with no pretreatment. Many of
the new & more esoteric antibodies require pretreatment. When possible, itıs
great to work up an antibody on both fresh & formalin fixed tissue. Well,
now my brain is beat. Hope this explains at least part of the reasoning
behind pretreatment.

Patti Loykasek
PhenoPath Laboratories
Seattle, WA




> Dear Histotechs;
> Placate an old retiree and get a quizzical question out of my head:  In
> 1974, I was assigned muscle and nerve biopsy work and immunofluorescence
> work on kidneys. Fixation ruined just about everything in all the
> procedures involved with those studies, so fixatives were OUT in all of
> my work. After moving to another hospital with my pathologist, I
> continued the muscle and nerve work but not the immunos. Over the years,
> news sort of trickled down that you histotechs were doing antibody work
> on fixed tissues.  I guessed you had found some way to repair the
> tissues after being fixed.  The Histonet has many messages sent back and
> forth between histotechs doing antibody work, and they always specified
> what fixative they used.  A nd that always bothered me.  It seemed like
> a ring-around-the-rosie to fix,  then,  Unfix tissues for antibody work.
> To up and fix the tissues, then turn around and repair SOME of the
> damage done to the tissues-because I thought the tissues would not come
> away unscathed from being bombarded  with a fixative. And if it was
> necessary to return the fixed tissues to an unfixed-like state, why not
> just leave them unfixed in the first place?  Does fixation do something
> good to the tissues, making them better for antibody studies than fresh
> frozen tissues? Muscle tissues are totally ruined for histochemistry,
> and there is no way to repair the harm done to them by fixation. It
> seemed like something out The Peterkin Papers:  One story from that
> wonderful book involved a cup of tea that had too much sugar in it.The
> lady who wanted to drink it went up and down the lane gettjng advice as
> to what to do, involved every herb, spice and remedy which just kept
> making the tea worse. But the little old lady at the end of the lane
> suggested that she make a fresh cup of tea. I wonder if the little old
> lady at the end of the lane had been consulted in this vase, she might
> have laughed merrily and suggested you just not fix your tissues in the
> first place. Is there is some improvement in the tissues brought about
> by fixation?  As fixation totally ruined all my work, I find it hard to
> believe that the tissues are going to come out unscathed from fixation.
> Anyway, this has been a mystery working in the back of my head for years
> .Can you put this matter to rest for this 76 year old?
> georgecole@ev1.net
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