[Histonet] Pink precipitate is monoformazan from BCIP/NBT reaction on ISH?

From:"Ralph Puchalski"

I am trying to figure out the origin of the pink precipitate in the
image I posted on http://www.histonet.org/site_images_frame.asp

Please go to the image entitled: Pink Precipitate ver2.jpg.  It is at
the top of the list on 9/26/05, posted by Ralph Puchalski.  To see the
pink precipitate artifact, please open the image and set the scroll bars
on the bottom and right side of the image at their 1/2 way points.  It
is ugly!

I think this precipitate is the aggregation of the monoformazan
intermediate generated from NBT (after dephosphorylation of BCIP by
alkaline phosphatase) that is not completely reduced to diformazan, the
insoluble dark blue or black precipitate that labels cells expressing
target mRNAs in our in situ hybridization reactions.  

We don't know how the monoformazan adheres to the sections of tissue.
It appears to be non-covalent due to the tendency of the monoformazan to
migrate under the coverslip in the aqueous mounting medium that has yet
to dry, and form clumps or aggregates or pools as seen in the picture.
The monoformazan is soluble in ethanol so doesn't form pools or
aggregates.  But as soon as it is exposed to an aqueous medium, it

If we mount the post-ISH tissue sections with an organic based mounting
medium like UV-CureMount (Instrumedics), the monoformazan might cause
the entire section to turn to a brown tint as seen on 

Please go to the image Brown Tint 1.jpg.  It is 4th image from the top
on 9/26, posted by Ralph Puchalski.   The lower image is mounted with UV
CureMount, and the upper was with aqueous Hydro Matrix.  There is no
pooling or precipitation of monoformazan with UV CureMount, but I think
it does cause the entire section to turn brown.

Question:  How do we eliminate this problem, which I believe is
monoformazan?  If we reduce it fully using ascorbate, the section (later
mounted with HyrdoMatrix) turns dark blue or purple, the same color as
our ISH signal.  We have tried washing off the monoformazan with 100%
ethanol prior to coverslipping, but only small amounts are removed.
100% acetone also does not work effectively.

Please let me know if you have any ideas that might help us eliminate
this problem.

Thank you,


Ralph Puchalski, Ph.D.
Manager, Process Engineering and Automation
Allen Institute for Brain Science
551 N. 34th Street, Suite 200
Seattle, WA  98103

Tel: 206-548-7041  Fax: 206-548-7071

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