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

[Peter van de Plas]

Dear Niall,

The effect on antigenicity when fixing biotin with aldehyde depends on 
the characteristics of the detection system you are using.

With the dot-spot test you have a easy approach to test this. Spot a 
dilution series of antigen (in your case a biotinylated protein) on a 
strips of nitrocellulose membrane and let dry. Individual strips are 
now incubated with the fixatives of interest, your control (reference) 
will be an unfixed strip. Wash in buffer and block free aldehydes in 
glycine. Remaining protein binding sites on the strip are blocked with 
BSA. Next step will be the incubation in your Streptavidin-HRP 
conjugate. Wash an develop the signal.

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In the past I did a comparable test for streptavidin-gold conjugates. I 
noticed a decrease in labeling density after fixation in paraform. 
There was only minor effect when using a polyclonal anti-biotin 
antibody.

Hope this info is of help.
Peter



On Thursday, May 27, 2004, at 04:28 PM, m. van mkempen wrote:

> 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)
>
>
-----------
Peter van de Plas
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