request: mouse mAB against mouse tissue??

Henk Veldman H.Veldman at lab.azu.nl
Mon Jun 14 04:06:20 EST 1999


Frank Dehner wrote:
> 
> Hi everybody,
> 
> how can I detect an antigen in mouse nervous tissue with a primary antibody
> raised in mouse (against rat protein)?
> I have ascites and supernant from cell culture and no idea about the Ig
> class of this AB.
> How can one avoid unspecific binding of the secondary antibody (which must
> be ....anti mouse) to the tissue?
> Any ideas about double labelling with a second mAB?
> Thanx a lot.
> Frank
> 
Just do the stain and you may be in for a surprise:
While in most tissues endogenous immunoglobulins are a big problem when
using autologous antibodies (or even higly crossreactive ones like mouse
and rat) in central nervous tissue this usually is not a problem. The
blood-brain barrier will have kept the immunoglobins out. Only blood
vessels will show and if you have used perfusion fixation even here most
of it will have washed away. However, when you are studying damaged
brain tissue, there might be a lot of immunoglobulin in the lesions. In
such cases you will have to resort to some form of prelabelling of your
antibodies, either by biotinylation or by preincubating with a secondary
antibody.

Good luck

Henk



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