RE: [Histonet] fixing immuno and antibody tissues

From:"Webster, Paul"

Hi George,

I think your questions will provoke more questions than answers. The problem is two-fold - good morphology with unlimited accessibility.
We want to preserve morphology so that we can immobilize antigens and localize them to specific, identifiable regions in the tissue, but we also want to give antibodies the maximum access to antigens. Sometimes these two aims cannot be met and often the end result will depend on the physical properties of the antigen. For example, if an antigen is embedded deep inside a membrane, then gaining access will require strong extraction treatments. Conversely, a soluble antigen will require stronger fixation to immobilize it in the tissue.

Unfixed tissues are usually immobilized by freezing. The frozen tissue is then sectioned, stuck to a slide and fixed, either by chemical fixation (aldehyde or acohol) or by drying. Sometimes it is a combination of these treatments. The reason why different treatments produce different results is because they offer varying degrees of accessibility.

It is interesting that your experience rules out fixatives because they "totally ruined your work". I guess that fixation treatments you performed were interfered with your antibody labeling in some way. However, in electron microscopy, chemical fixation is almost always a prerequisite for immunolabeling. Many antigens survive embedding in acrylic or epoxy resins. Similarly, there are many enzymes that remain active after aldehyde treatment and form the basis of the very successful field of EM cytochemistry. I suspect that antibody production technology has greatly improved over the last 40 years and may be one reason why immunolabeling has become more versatile and successful, even on fixed tissues.

It is interesting that you perceive fixation as doing damage and then the subsequent treatments, which often involve boiling sections in citrate buffer in a pressure cooker (!), as repairing the damage done by the fixative.  I would suspect that a good fixation (e.g. in phosphate-buffered formaldehyde) will immobilize most proteins in a tissue and form cross-links between the proteins that may interfere with antibody access to antigens. Only the subsequent hydrolysis that damages the tissue section and which is often called antigen retrieval, is able to restore the access to antigens.

Using unfixed tissues may offer easy access to antigens but then you run the risk of antigen migration if there is no immobilization treatment. This may not be a problem in histology where tissue sections are examined at relatively low magnification, but it is an important problem in electron microscopy, where antigens moving distances of less than 1Ám can produce meaningless results.

This brings me to a question of my own. 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

Disclaimer: I am an electron microscopist who has been hanging out here because it currently seems more fun than the MSA listserver.

> ----------
> From: on behalf of George Cole
> Sent: 	Tuesday, August 10, 2004 12:28 PM
> To:
> 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?
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