Re: periodate-lysine-paraformaldehyde fixative
From: | Paul Klosen <klosen@neurochem.u-strasbg.fr> |
At 12:24 20/06/01 -0700, Ford, Judi {Path~Palo Alto} a écrit:
>Hi everyone!
>
>I was wondering if anyone has a detailed recipe for PLP fixative. One of my
>clients wants to use this fixative for
>their immuno work-up and we don't have anything on it in our lab.
Dissolve 7.3 g of lysine monohydrochloride in 200 ml of ddH2O. Adjust pH to=20
7.4 with 0.1 M Na2HPO4 (Na2HPO4 . 2 H2O 17.8 g/l) NOT phosphate buffer
!!!. Complete volume to 400 ml with 0.1 M phosphate BUFFER (!!) pH 7.4.
This lysine-phosphate buffer keeps for 2-3 days in the refrigerator, but
can be frozen in aliquots for longer storage.
Just before use, mix 375 ml lysine-phosphate buffer with 100 ml 20%
Formaldehyde and top to 500 ml with ddH2O. Add 1.06 g NaIO4 and mix well.
The PLP fixative must be used within maximum 2 hours.
Final concentrations: Lysine 75 mM, Formaldehyde 4%, Sodium periodate 10 mM
The original PLP fixative by Nakane & co used 2% Formaldehyde and a
variable amount of periodate. This is the mix that works best for us for
both ICC and ISH.
Paul
-=3D-
(o -) O
==========================3D=====oOo==(_)==OOo=============3D================
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. 03.90.24.05.01 Fax. 03.90.24.05.28
========================klosen@neurochem.u-strasbg.fr==================3D=======
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