RE: Hirschpurng's for J.A. Kiernan

From:Stephanie Moore <>

Dr. Kiernan,

I noticed that your newest book has an updated AChE protocol.  I have
ordered this book through our university library, however it has not yet
arrived.  Before I try this, I just want to verify something.

You show both a full strength and 10x dilution of the incubation medium
and inform that full strength is for use with tissues containing high
enzymatic activity while the diluted medium is for tissues with low
enzymatic activity or AChE-positive nerve fibres.  This seems
counterintuitive and I was wondering why this works.  I have a staining
coming up and would like to do it the best way possible.  I have been
using "full strength" incubation medium for my sqirrrel brain sections. 
Additionally, the previous method used DAB (w/out peroxide) and .01%
osmium tetroxide to enhance staining.  I found that my slices came out
VERY dark!  This "newer" method has no osmium tetroxide step in the
enhancement and adds the more common reagents associated with DAB:
peroxide and nickel ammonium sulfate (and possible/probable gray
background).  I assume that the more recent enhancement method is
better?  Any way to reduce background?

Summary questions:
Should I use the diluted medium for my brain sections?
Which enhancement method works best?

Thank you so much - I always find you helpful :)

Stephanie Moore

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