Re: Glutaraldehyde

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From:"J. A. Kiernan" <> (by way of histonet)
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BOn Fri, 10 Dec 1999, Colleen Forster wrote:

> Again I have a fixative question. What is the difference between
> paraformeldehyde and glutaraldehyde, chemically speaking. I recently
> used a fixative that was part of each for an EM project and noticed ...
> I am sure that John Kiernan among others will be able to help me out.

Oh, all right then :(
It can't be done briefly. You should persuade your boss
to invest $300 of the lab's budget in 5 or 6 textbooks
and a small shelf to keep them on, above a 50 cm length
of bench.

  Formaldehyde has small molecules: HCHO, of which the -CHO is the
  aldehyde group that can combine with nitrogen atoms of proteins
  (or with two such N atoms if they are very close together, forming
  a crosslink -CH2- called a methylene bridge. Paraformaldehyde is
  an insoluble polymer of formaldehyde. It becomes formaldehyde
  monomer when "dissolved" in buffer.

    Now the important bit: Formaldehyde penetrates tissue quickly
    (small molecules), but its reactions with protein, especially
    cross-linking, occur slowly.  Fixation takes days, especially
    if the specimen must withstand the osmotic and other stresses
    of dehydration and paraffin. Brief fixation in formaldehyde
    (ideally delivered by perfusion) can stop or greatly reduce
    autolysis and confer slight hardening and some resistance (but
    not much) to liquides that are not iso-osmotic with the tissue.
    This can greatly improve the structural integrity of cryostat
    and other frozen sections, especially if followed by infiltration
    with a cryoprotectant such as sucrose (ideally 60% but more
    usually 15-30%).

  Glutaraldehyde has fairly small molecules, each with two aldehyde
  groups, separated by a flexible chain of 3 methylene bridges. It
  is HCO-(CH2)3-CHO. The potential for cross-linking is obviously
  much greater because this can occur through both the -CHO groups
  and over variable distances. In aqueous solutions, glutaraldehyde
  is present largely as polymers of variable size. There is a free
  aldehyde group sticking out of the side of each unit of the
  polymer molecule, as well as one at each end. All these -CHO groups
  will combine with protein nitrogens with which they come into
  contact, so there is enormous potential for cross-linking, and that
  is just what you get. There are also lots of left-over aldehyde
  groups (not bound to anything) that cannot be washed out of the

  There are five important bottom lines for glutaraldehyde.

    1. If it's to be any good as a fixative, especially for EM, the
       glutaraldehyde solution must contain the monomer and low
       polymers (oligomers) with molecules small enough to penetrate
       the tissue fairly quickly. This means you must buy "EM grade"
       glutaraldehyde (25% or 50% solution), not a cheaper "technical"
       grade. The cheaper stuff is for tanning leather. A tanner's
       son told me that only the best and most expensive leather goods
       are tanned in glutaraldehyde.
    2. The chemical reaction of glutartaldehyde with protein is fast
       (minutes to hours), but the larger molecules, especially the
       oligomers, penetrate tissue slowly. (A rat's brain left
       overnight in a buffered glutaraldehyde solution and sliced
       the next day shows a colour change and harder consistency
       to a depth of 2-3 mm.) Objects fixed for a few hours in
       glutaraldehyde are no longer osmotically responsive.
    3. The free aldehyde groups introduced by glutaraldehyde fixation
       cause various problems (non-specific binding of proteins, such
       as antibodies; direct reaction with Schiff's reagent etc etc).
       These can all be overcome by appropriate histochemical
       blocking procedures, as described in textbooks.
    4. The thorough cross-linking of a glutaraldehyde-fixed specimen
       impedes the penetration of fairly large paraffin wax molecules.
       This makes for difficult cutting and peculiar differential
       shrinkage artifacts within the specimen. You can stain
       mitochondria nicely in cells surrounded by obviously abnormal
       spaces. This is an exaggeration of the inadequacy of
       formaldehyde and osmium tetroxide as fixatives to precede
       paraffin, and it also highlights the shortcomings of
       predominantly coagulant fixatives (AFA, Davidson's, Bouin etc),
       which preserve the micro-anatomy well but destroy or displace
       little things like organelles.
    5. Immunohistochemistry, which requires as many intact amino acid
       side-chains as possible, is severely impaired by glutaraldehyde
       fixation. Nevertheless, clever people have generated antibodies
       to individual, "free" but glutaraldehyde-bound-to protein amino
       acids. These allow the detection of soluble amino acids such as
       glutamate, GABA and even glycine in presynaptic axon terminals
       - in glutaraldehyde-perfused CNS.

  The combination of formaldehyde with glutaraldehyde as a fixative
  for electron microscopy takes advantage of the rapid penetration
  of HCHO and the thorough cross-linking by glutaraldehyde. The
  credit is nearly always given to M. Karnovsky of Boston, MA, who
  published such a mixture in an unrefereed abstract in 1965. His
  mixture contained more glutaraldehyde than most people came to like,
  and designations like "half-strength Karnovsky" became common
  parlance. Fixatives of this kind allowed the definitive descriptions
  of EM-level histology that were largely completed in the following
  5 or 6 years. Details and amendments continue to be added.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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