NADPHd artifact

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From:Susan Travers <>
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I am writing to ask if anyone has seen and has a solution to an artifact 
that we have seen in our NADPHd material.  It appears as a small, elongated 
crystalline precipitate that appears to be on the surface of the tissue. 
Our technique for doing the NADPHd is the standard one using .1% bNADPH & 
.01% nitroblue tetrazolium in PB. The only twists are: that the tissue is 
fixed with PLP , and we double-label the tissue using DAB 
immunohistochemistry (first NADPHd, then ICC)

We have tried to trouble shoot by (1) filtering the staining solution-- no 
help, (2) doing the reaction in the dark (inconvenient and no help), (3) 
exploring the effects of using ETOH to dehydrate or not-- this is 
interesting-- with ETOH, which is our standard procedure the artifact looks 
crystalline, without ETOH-- looks like very small spherical globules, (4) 
using xylene or HEMO-D to clear-- no effect, and (5)coverslipping with 
permount versus cytoseal (no effect).

The only other hints are that we never see the artifact on wet, 
uncoverslipped sections, and occasionally it seems that the artifact gets 
worse the first few days after coverslipping; also the artifact does seem 
worse in the double-labeled tissue.

Any advice would be greatly appreciated.

Susan Travers 

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