[Histonet] RE: alk phos, again

[C.M. van der Loos]

   Hi Rachael,

   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
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   phone:  +31 20 5665631
   fax:    +31 20 6960389
   e-mail: [1]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.......
   Thanks!
    Rachael

References

   1. mailto:c.m.vanderloos <@t> amc.uva.nl