Re: Fixative Buffering
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From: | Paul Klosen <klosen@neurochem.u-strasbg.fr> |
To: | "Jacques Paysan" <paysan@uni-hohenheim.de>, "HistoNet Server" <histonet@pathology.swmed.edu> |
Reply-To: | |
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Hi,
Well it looks like molecular biologists are discovering a few things that
histologists and immunocytochemists learned a few years ago.
Alkaline fixation using formaldehyde has been described for ICC in the
80's. One paper that come's to mind is the work of Anne Berod and coworkers
(1981) in JHC 29 (7): 844-850.I used these fixa tive
The fixative used by these workers was 4% formaldehyde in Borate buffer pH
11. I know, pH11 is just outside the buffering range of most borate
buffers, but it worked. For perfusion fixation, the alkaline formaldehyde
fixatives are preceded by neutral formaldehyde. At alkaline pH, the amount
of formaldehyde in the aldehyde form is increased. The fast penetration of
formaldehyde (as compared to glutaraldehyde) however stems from the fact
that most of the formaldehyde is in enol form at neutral pH. The enol form
permeates cell membranes much faster than the aldehyde form. Thus, for
perfusion fixation you first saturate the tissue with formaldehyde at
neutral pH, then you raise the pH for the rest of the fixation.
I used this fixative for several years for ICC, where I got very good
results. When doing ISH, I noticed that you had to use more proteinase K to
obtain good signal to noise ratios. We switched to PLP
(Periodate-Lysine-Paraformaldehyde) fixative a few years ago because of its
superior results for ICC, while maintaining the quality for ISH. But we add
0.04% DEPC in the PBS perfusion rinse, which has been shown to increase ISH
signals.
As to the buffer substances you might use for alkaline pH, most buffers
above pH 9 contain borate (Clark & Lubs, etc) or carbonate. There also is
the Glycine-NaOH buffer, but which probably is not adequate. Finally,
phosphate can be used between pH 11 and 11.9.
For other buffer substances, you can check in the Sigma catalogue in the
Biological buffers section. I see CHESand CAPS in the 9-10 and 10-11 range,
but I don't know how these behave in the presence of formaldehyde.
Paul
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(o -) O
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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur 12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04 Fax. 03.88.24.04.61
========================klosen@neurochem.u-strasbg.fr=========================
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