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

[m. van mkempen]

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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