Re: Fwd: ALP for histonet

From:"J. A. Kiernan"

On Thu, 4 Oct 2001, John Baker wrote:
> Hi all,  This message is from a graduate student in our laboratory. 

Advise her to look up the 2nd or 3rd (not the 4th) edition
of Pearse's Histochemistry.

Activity is seen in frozen but not in paraffin sections,
so processing and/or infiltration must be what's wrecking
the enzyme. Pearse gave instructions for decalcifying,
dehydrating and embedding in low temperature wax, complete
with references to the original papers by George Gomori
and others who developed this technology in the 1940s and
early '50s. She'll need primary sources if the work will
end up as an MSc or PhD thesis.

For simple alkaline phosphatase histochemistry it shouldn't
make much difference which substrate and trapping reaction
she uses. They'll all show the enzyme if it's there and 
active. Exact localization within cells may not be the
same with all techniques because of diffusion of intermediate
reaction products, but that's another story - and one that
has to be really thoroughly understood by every graduate
student who does enzyme histochemistry and wants to survive
the viva. 

> >out the Alkaline Phosphatase staining protocol ...
> >have been attempting to get ALP staining on paraffin sections of
> >decalcified trabecular bone.
> >Thanks!   Nancy Caldwell

For direct practical advice: You're in Nottingham! Is John Bancroft
still there too? He should have the answers, and they may also be
in his earlier books, which are practical and much smaller than
Pearse, but also less academic.

Wouldn't it be wonderful if something like heat induced
antigen retrieval would re-activate enzymes in paraffin
sections!

----------------------------------------
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1
   kiernan@uwo.ca
   http://publish.uwo.ca/~jkiernan





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