RE: Alkaline phosphatase

From:Dan Beniker

Woo Hoo!!! I've been wrestling with that very beast for some time!!!! I've
heard many reports that it's possible and even routine in some labs, but we
weren't able to repeat other's success here....for a while. What I've been
able to pin down is that the ability to stain for AP in paraffin is
dependant on several variables... Assuming you're looking for AP in bone,
the method of decal is very important. What I've found is that EDTA, while
considerably slower, is the decal reagant of choice for preserving enzymatic
activity. Formic acid (or any acidic decal)in my hands destroys activity. I
also try to limit extreme temps in processing. We do decals at 4 deg. C and
lower the temp of the paraffin until it's just above it's melt point. In our
lab that was about a 5 degree drop in temp from what we normally run things
at. Also, I'm somewhat biased towards fixatives... Although I haven't
verified this, I personally feel that the Zinc Formalin fixatives add a
little bit to preservation. By adjusting these two parameters we have been
able to get decent AP staining in bone in paraffin embedded sections... The
down side so far is that it takes a while to see the results!

I'm currently trying a shortened EDTA decal in elevated temps (25 and 37
deg. C) in the hopes that it will speed decal and preserve activity...Sounds
counterintuitive, but I figure that, hey, the enzyme normally functions at
those temps, so it might be worth a shot! 

> -----Original Message-----
> From:	Karen M Majahad []
> Sent:	Friday, May 03, 2002 1:45 PM
> To:
> Subject:	Alkaline phosphatase
> Has anyone had any luck with staining for alkaline
> phosphotase in paraffin sections. I was told that it is very
> hard to do enzyme staining on paraffin sections.

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