biotin binding to avidin after fixation?

dd dd
Mon Apr 1 08:36:06 EST 1996


In article <v02130500ad81aa97aa23@[134.76.211.74]>,
jgoed at MPC186.MPIBPC.GWDG.DE (Joachim Goedhart) wrote:

> Hallo,
> 
> As a student I'm now using the ABC (biotin-EGF, avidin, biotin-peroxydase)
> system to label EGF receptors on  cells. However, we have the problem that
> avidin enters cells that are fixed with 3.7% PFA. To avoid this, we want to
> fix the cells after adding avidin. This is probably the only way, because
> fixation after adding biotin-peroxydase will inactivate the enzyme. Does
> anybody know if the biotin-binding sites will be destroyed by PFA?
> 
> Thanks,
> Joachim Goedhart

I think you are incorrect in assuming PF fixation will inactivate
peroxidase.  If you have done peroxidase staining in the past in mammalian
tissues, you may have noted the high background in cells with endogenous
peroxidases (e.g., erythrocytes).  I have published a paper in which we
perfused rats with HRP, then fixed with glutaraldehyde/formaldehyde, then
diced the tissue, ran the peroxidase reaction, fixed with osmium, embedded
in resin, and thin sectioned for EM.  we got great localization (lots of
others have done this also).  see Cell Tissue Res (1987) 247:547-554. 
good luck.



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