Re: fixative question for chemistry experts

<< Previous Message | Next Message >> (Paul Klosen)
To:Marcia Bentz <>
Date:Sat, 04 Sep 1999 01:18:04 +0200
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>Hi all,
>Just wondering if anyone out there's heard of a fixative called PLP.
>It contains:
>4% paraformaldehyde dissolved in dH2O plus 2 drops of 10M NaOH
>sodium m-periodate (NaIO4)
>sodium phosphate. I'm not sure of the concentrations, I have a recipe for a
>given amount of solution.

We use a modified PLP fixative with a final concentration of 4%
formaldehyde, 75 mM Lysine-HCl and 10 mM sodium metaperiodate in phosphate
To prepare the lysine-phosphate buffer, start with 1 volume 200 mM
Lysine-HCl solution and titrate the pH to 7.2-7.4 with 100 mM Na2HPO4.
Complete to 2 volumes with 100 mM phosphate buffer pH 7.2-7.4. This buffer
keeps only for one or to days, but can be frozen in aliquots for longer
The fixative should be prepared just before use.
Combine 1 volume 16% formaldehyde with 3 volumes lysine-phosphate buffer
and add NaIO4 to 10 mM.

This fixative was initially designed by Paul Nakane. The rationale behind
this complicated mix is that the NaIO4 should oxidize trans-diol containing
hexoses to Schiff bases. These in turn should react with one of the amino
groups of the lysine, the other amino group reacting either with another
Schiff base or the formaldehyde. The fixative design is obviously targetted
at glycoproteins. It has also been used to perform deoxyglucose activity
measurements at the cellular level (McCasland & Woolsey 1988, J Comp Neurol
278: 543-554).

>My questions are:
>Is this approximately the same as dissolving 4% paraformaldehyde in PBS?
>What is the difference between this and 10% buffered formalin?

This fixative is not quite comparable to buffered formalin. In our hands,
it penetrates very poorly into tissue blocks by immersion fixation. We
tried it on muscle and nerve biopsies for paraffin embedding, and the
morphology turned out very bad. However, on perfusion fixed research
material the results were very satisfactory. Actually it has become our
standard fixative for ICC and ISH on fixed-cryoprotected-frozen sections
and on PEG (carbowax embedded material). All antigens that work on
formaldehyde-fixed material work at least equally well on PLP fixed
material. Mostly they work even much better, especially structural antigens
like intermediate filaments. One neurofilament clone, NE52 against
non-phosphorylated NF-H worked barely on formaldehyde-fixed tissue at a
dilution of 1/200. On PLP fixed material, we increased the dilution to
1/600-1/1000 and still obtained a very good staining of both neuronal cell
bodies and axons. We have obtained similar results with antibodies against
MAPs and macrophage antigens (the ED series).

Paul Klosen

                         (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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