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|From:||"J. A. Kiernan" <firstname.lastname@example.org> (by way of histonet)|
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
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
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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