[Histonet] fixing immuno and antibody tissues

Gayle Callis gcallis <@t> montana.edu
Wed Aug 11 11:22:39 CDT 2004


In response to your last question about smaller pieces of tissue: Jerry 
Fredenburgh (Richard Allan) gave an explanation (chemistry is one of his 
hobbies!) that bound water on protein can be removed along with free water 
found in tissue spaces.  The object is to retain bound water, and only 
remove the free water.   If you overexpose to alcohol during dehydration or 
add heat to processing during dehydration steps, bound water is removed, 
tissue becomes more brittle, dry, difficult to section.  I think you 
answered your own question with your last sentence, although clearing 
agent, e.g. xylene is there to remove alcohol in preparation for 
infiltration by paraffin and/or some plastics (methylmethacrylate).  Xylene 
is considered hardening to tissues also, more so than some of the xylene 
substitutes, eg Propar and Clearite 3 - single aliphatic 
hydrocarbons.  Heat during paraffin infiltration also dries tissue and 
contributes to brittleness (many of us have had paraffin bath temperature 
failures, and the tissue is "cooked" into rockhard nuggets when the 
paraffin is too hot.

Just some thoughts - also, in our work with murine CD marker 
immunohistochemistry, any aldehyde fixative creates chaos, and 
immnuonstaining is never achieved even with minimal fixation time, or after 
any kind of enzyme digestion and retrieval method - CD4 and CD8 are 
notorious (how many times have I said this- ad nauseum!?) .  We then rely 
on fresh tissue frozen sections fixed after air drying and with acetone 
alcohol mixture OR acetone fixation) precipitating these antigens rather 
than aldehyde crosslinkage.   Maybe some day there will be CD4 and 
CD8clones that work with FFPE mouse tissue ----- waiting --------------

At 06:06 PM 8/10/2004, you wrote:

>If anyone is stillrreading, maybe they could offer and explanation of why 
>histologists use shorter processing times for smaller tissue blocks. I 
>have been reading the recent thread about smaller blocks being brittle 
>because they were being processed for too long a time. Does anyone know 
>why the tissue becomes brittle? I am going to stick out my neck and say it 
>is probably not because of over-fixation in formaldehyde because I 
>routinely cut thin sections (100nm) of tissues that have been left in 
>formaldehyde for weeks. The tissues are infiltrated with sucrose, frozen 
>and sectioned at -120 degrees and there is no difference between them and 
>tissues fixed for only a few hours. Could the problem with brittle blocks 
>be a result of exposure to alcohol or xylene?
>Thanks for your time.
>Paul Webster.
>Paul Webster, Ph.D.
>House Ear Institute
>2100 West Third Street
>Los Angeles
>CA 90057
>phone (213) 273 8026
>fax      (213) 413 6739
>email: pwebster <@t> hei.org
>Disclaimer: I am an electron microscopist who has been hanging out here 
>because it currently seems more fun than the MSA listserver.
> > ----------
> > From:         histonet-bounces <@t> lists.utsouthwestern.edu on behalf of 
> George Cole
> > Sent:         Tuesday, August 10, 2004 12:28 PM
> > To:   histonet <@t> lists.utsouthwestern.edu
> > Subject:      [Histonet] fixing immuno and antibody tissues
> >
> > 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 <@t> ev1.net
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> >
> >
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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