Re: [Histonet] NFB vs. Paraformaldehyde
This question comes up frequently on Histonet.
The simple fact is that paraformaldehyde is an
insoluble solid polymer that depolymerizes when
heated in buffered water. The solution you end up with
contains no paraformaldehyde at all, and is almost
identical to a buffered formaldehyde solution made
from formalin (which is a 37% solution of formaldehyde).
Formalin consists largely of smaller polymers, which
break down on dilution with water or buffer. A fixative
made from formalin contains about 1% methanol, which is
not present when paraformaldehyde is the source of the
formaldehyde. If a formalin stock is very old (years)
there will be more methanol, and also some formate ions.
Small amounts of methanol and formate do not affect the
fixative properties. A properly formulated buffered
formaldehyde solution, made from formalin, is good even
for subsequent electron microscopy. It is more trouble
to make the fixative from paraformaldehyde.
Remember, nobody has ever fixed a specimen in
paraformaldehyde; this substance exists only in
the solid state and is insoluble. Old bottles of
formalin often contain a white precipitate in the
bottom; this is paraformaldehyde. Methanol (about
10%) is added to formalin to retard the formation
of this high polymer. For longer essays on this
topic, look up:
Researchers these days frequently do not know the
basics of fixation, staining or microscopy. For
them, there is:
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
> Good Morning Histonetters,
> I would like to get an idea from those histotechs that work with mouse tissue if
> they prefer NFB or Paraformaldehyde as a fixative for routine histology.
> I work for a group of 6 researchers, most of who do mouse work. Their students
> do the necropsies, remove and block the tissue, make up their own
> paraformaldehyde, fix and submit the cassettes to me from processing and
> I would like everyone to change to NFB to standarize the fixation. I can
> guarentee that no one makes Paraformaldehyde the same. Some make it the same
> day, some the day before, 2-3 days before etc.
> I am meeting with the big bosses this afternoon and would like some ammunition
> if possible.
> Thanks for the assistance.
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