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

[John Kiernan]

Dear Ralph,

Your email is full of abbreviations and jargon. 
Am I right in thinking it's a question about 
localizing alkaline phosphatase activity by an
indoxyl-tetrazolium method? If so, please provide
a reference to the original publication and let us 
all know if you followed it exactly or made any
changes. Part of your email suggests that the
alkaline phosphatase activity is not endogenous 
but part of an amplification system used in
in situ hybridization. The pH optima of endogenous
and label alkaline phosphatases differ.

The later paragraphs of your message indicate that
you may not fully understand the significance of
the mono- and diformazan products of reduction of 
nitro-BT. Both colours result from reduction of
the tetrazolium salt, but you need controls to 
prove that the reduction was by bromochloroindoxyl
and not by other reducing agents (such as diaphorases)
in the tissue. Non-enzymatic reduction of tetrazolium
salts by -SH has been a well known artifact {"nothing
dehydrogenase") for 40-45 years. 

John A. Kiernan
Anatomy Dept, UWO
London, Canada.
___________________________________________________
Ralph Puchalski wrote:
> 
> 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
> precipitates.
> 
> 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
> http://www.histonet.org/site_images_frame.asp
> 
> 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
> 
> Ralph Puchalski, Ph.D.
> Manager, Process Engineering and Automation
> Allen Institute for Brain Science
> 551 N. 34th Street, Suite 200
> Seattle, WA  98103
> 
> ralphpu <@t> alleninstitute.org
> Tel: 206-548-7041  Fax: 206-548-7071
> www.brainatlas.org
> 
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