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

From:"Ralph Puchalski" (by way of histonet)

 Dear John,

Thank you for your advice.  Yes, we use digoxigenin (DIG)-labeled RNA
probes (riboprobes) in the in situ hybridization of frozen sections to
map gene expression throughout the mouse brain.  Detection of bound
riboprobe is a multi-step procedure.  First, a succession of blocking
steps inhibits endogenous protein activity from interfering with the
colorimetric enzymatic reactions.  The colorimetric reaction itself is a
four part process, starting with the addition of a peroxidase-conjugated
antibody directed at the DIG-UTP hapten incorporated in the bound
riboprobe.  A Tyramide Signal Amplification step is utilized to maximize
sensitivity.  In brief, biotin-coupled tyramide is added to the tissue,
resulting in the formation of multiple activated tyramide molecules
through the activity of each bound peroxidase-coupled antibody molecule.
These highly reactive tyramide radicals bind to protein residues in the
vicinity of the bound riboprobe, thereby resulting in an amplification
of bound biotin molecules available for detection by up to a hundred
fold (relative to the number of bound antibody molecules).  These biotin
molecules are then bound to NeutrAvidin-AP, the third step of the
colorimetric reaction.  Alkaline phosphatase (AP) conjugated to the
NeutrAvidin (NA-AP) enzymatically cleaves the phosphate from
5-bromo-4-chloro-3-indolyl phosphate (BCIP), and two of the resulting
indoles undergo a redox reaction with nitroblue tetrazolium (NBT) to
produce a blue particulate precipitate at the sites of riboprobe
binding.  We process about 800 slides (1600 sections per day), and you
can visit our site for more details of our method and product at:;jsessionid=9

Processing 800 slides per day in order to cover the entire genome of
about 20,000 genes necessitates the use of robots.  The slides are
incubated in only 300 ul of a particular reagent solution in chambers
mounted on an angle.  The incubation periods and concentrations were
optimized for detection of the lowest levels of target RNA expression.
The conditions were established for the entire project and major changes
cannot be made.  The system was set up so that all sections receive the
same amount of all reagents, including BCIP, NBT, and NA-AP.

You commented on nothing dehydrogenases.  Our experiments demonstrate
that removing NA-AP from the reaction, while keeping all other steps as
they are in the control, produces sections that are transparent, i.e.
without blue or purple staining under bright-field microscopy.  If
anything, the sections have a very faint yellow tint, the same as what
we observe in the bottles just after we prepare the BCIP/NBT substrate
in buffer with Levamisole.  However, if we incubate these slides in a
reducing agent such as 50 mM alkaline ascorbate for 5 minutes in pH 9.5
in Tris-Magnesium-Saline, the slides paired to the ones that are
transparent become stained deep purple-blue (no NA-AP).  Therefore, if
we are interpreting the data correctly, nothing dehydrogenases
contribute nothing that is detectable, and ascorbate reduces NBT to
diformazan.  Do you agree?

The troubling point is that the amount of staining visualized by
reduction with ascorbate matches the staining by the pink precipitate
(formed in the presence of NA-AP in our production runs and controls),
which presumably is monoformazan as determined spectrophotometrically.
The staining levels are highest in the fiber tracts, brainstem, and
hypothalamus, but are visible throughout the sections to various
extents.  The reason why this is troubling is that the staining does not
require NA-AP.  The NBT (we will test to be sure BCIP is not required)
just sticks to these areas, and later ends up on production slides
(NA-AP+) as monoformazan or pink precipitate, which is our hypothesis.
Do you agree with this assessment?

How would you go about decreasing the staining of the fiber tracts,
brainstem, and hypothalamus, and the rest of the section with NBT?  We
plan to titrate the NBT, but cannot reduce signal levels.  Our fixation
and dehydration process does not remove all the fats in the tissue, so
it is quite possible that the hydrophobic NBT gets partitioned into the
lipid phase.  Would you attempt to change the BCIP/NBT/Levamisole buffer
(Tris-HCl, NaCl, and MgCl2, Tween 20) used in the incubation with NA-AP
for color development?

Do you know if anyone has tried to block the endogenous sites to which
NBT adheres?  Without knowing how the binding occurs, it is difficult to
suggest options.  How would you go about characterizing the binding of
NBT to the sections?  I wish it were as easy as finding a convenient
analogue of NBT that is colorless, does not inhibit AP activity, is not
reduced, and binds to the same sites as NBT does, thereby blocking NBT
>from binding to these sites.  Any suggestions?

Your advice is appreciated.

Ralph Puchalski

-----Original Message-----
From: John Kiernan []
Sent: Monday, September 26, 2005 9:42 PM
To: Ralph Puchalski
Subject: Re: [Histonet] Pink precipitate is monoformazan from BCIP/NBT
reaction onISH?

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
> 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
> 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,
> 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
> migrate under the coverslip in the aqueous mounting medium that has
> 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
> 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
> CureMount, and the upper was with aqueous Hydro Matrix.  There is no
> pooling or precipitation of monoformazan with UV CureMount, but I
> 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
> 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
> Tel: 206-548-7041  Fax: 206-548-7071
> _______________________________________________
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