Re: [Histonet] Is biotin "inactivated" by fixation with paraformaldehyde?

From:Todd Sherman

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Dear Niall,

I'm not familiar with the detection system you have described, i.e.
membrane-impermeant HBSS/Sulfo-Biotin-X-NHS (Excuse my ignorance but
what is HBSS?) but the incubation time and temperature seems extremely
brief? Is that what the product insert recommends or what some other lab
has used successfully? Have you had success with this previously? Is
+4deg C the optimum temperature of reactivity? The lung temperature will
be elevated for a while until the reagent gets flushed through the
entire system and equilibrates. Do you begin timing as soon as reagents
enter the trachea or when the reagent has had a chance to reach the most
terminal bronchioles? Did you detect any "staining" at the most proximal
levels, i.e. sections where the reagent had the most time to bind, or
were bronchus to terminal bronchioles equally devoid of DAB complex?

Or another question: Were pulmonary secretions (mucous) flushed out
prior to incubation with HBSS/Sulfo-Biotin-X-NHS? Maybe the secretions
served as a physical barrier to your incubate.

I know I'm not specifically answering your aldehyde issue but these
other issues pop into my head first. You'll just have to excuse my

Now as long as there is free amine, then the aldehyde can bind and, I
guess, deter subsequent binding by streptavidin-HRP. So if the
Sulfo-Biotin-X-NHS has free unbound amine, then methylene-bridge
formation will continue. I just don't know the binding sequence of the
reagents involved. Is this the theoretical model?

~                           |
~                           v
~                      Streptavidin
~                           |
~                          HRP
~                           |
~                           v
~                          DAB

Which is the "lumenal end" of the Sulfo-Biotin-X-NHS complex? What does
HBSS bind to?

Hope this helps,

Todd Sherman
HistoSoft Corporation
"Biology in a new form..."
Member Services: wrote:

| Today's Topics:

|   23. Is biotin "inactivated" by fixation with	paraformaldehyde?
|       (m. van mkempen)
| ----------------------------------------------------------------------
Message: 23
Date: Thu, 27 May 2004 16:28:08 +0200
From: "m. van mkempen" 
Subject: [Histonet] Is biotin "inactivated" by fixation with
To: Histonetlist 
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Hello everybody,

I have a question about biotin and fixation in paraformaldehyde.

The experiment is as follows:

Intact lungs from adult mice were labeld with HBSS containing
Sulfo-Biotin-X-NHS. The solution was injected through the trachea and
the labeling took 15min at 4 degrees Celcius

Afterwards the lungs were flushed with PBS/glycin to remove any
unreacted biotin

The lungs were fixed in 4% paraformaldehyde for 48h

Tissue was embedded in parafin and cut into 4 micron sections

I used DAKO ABC (streptavidin-HRP) and DAB to detect the biotin labeled
proteins. Since Sulfo-Biotin-X-NHS is water soluble I would expect to
see a small line of labeled proteins on top of the lung epithelium.

But after a few tries I am unable to detect anything. Should I use HIER,
switch completely to cryosections or...?

One the internet I read that endogenous biotin is partly inactivated by
formalin fixation. Could this explain the problems I have?

Best regards, Niall Oudesluys (ErasmusMC, Rotterdam, The Netherlands)

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