[Histonet] RE: alk phos, again
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Thu Feb 24 02:44:19 CST 2005
You wrote that you consider the observed "background" as endogenous
alk. phos. activity. Please realize that real endogenous alk. phos.
activity shows up specifically in vessels for example rather than an
overall non-specific background staining. The fact that high
concentrations of acetic acid did kill your background doesn't say
anything as it also killed your specific signal.
The Dako Dual Endogenous Enzyme Block reagent (S2003) blocks both
endogenous peroxidase and alk. phosphatase activities. However, as I
wrote on Histonet before it may affect your epitopes as well.
There is another solution, a quite mysterious one I should admit but
it works good. It's both a fixative and blocking reagent
for endogenous peroxidase and alk. phosphatase activities. Prepare 4%
sodium nitrite and 4% pararosaniline solutions and mix 1:1 for exactly
1 minute under the fume hood. Than, mix 2 ml of diazotized
pararosaniline with 200 ml distilled water and store at 4C in the
dark. Fix your cryosections for exactly 2 min in cold fixative and
wash with TBS or PBS. Ref: Schrijver et al. JHC 48:95-103, 2000.
Again, after using this fixative/blocking reagent you have to check
whether or not your epitopes are affected or not.
Hope this helps,
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
e-mail: c.m.vanderloos <@t> amc.uva.nl
----- Original Message -----
From "Emerson, Rachael" <Rachael_Emerson <@t> URMC.Rochester.edu>
Date Tue, 22 Feb 2005 14:12:56 -0500
To "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Subject [Histonet] alk phos, again
Other there other methods to block endogenous alkaline phosphatase?
I am using mouse embryos, frozen sectioned. I've tried:
levamisole at varying concentrations: still background
heated PBS at varying times: too strong, blocked my signal
15% acetic acid at 15": too strong, blocked my signal
5% acetic acid at 15": still background
usually I did a combo of levamisole and another treatment.
I appreciate any thoughts.......
1. mailto:c.m.vanderloos <@t> amc.uva.nl
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