Re: Fixative Buffering

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From:Paul Klosen <>
To:"Jacques Paysan" <>, "HistoNet Server" <>
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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 

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.

                                                                        (o -) O
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.  Fax.

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