"> -----Original Message-----

> > From: Tibor Ric [SMTP:tibor@home.se]
> > Sent: Wednesday, April 12, 2000 12:49 PM
> > To:   HistoNet Server
> > Subject:      anti-calretinin and pale brown DAB reaction
> >
> > Hi everybody!
> >
> > I'm using anti- calretinin from Swant (Code No 7699/4, polyclonal) in my
> > project. The problem is that I'm getting too pale DAB reaction. I did
> > everything like before for HBME-1 and CK 5/6 and they were not that pale
> > brown. I'm sure that the DAB solution is okay. The material I'm using is
> > from 1991 to 1999.
> >
> > One problem I consider here would be the fixation time which I don't know
> > for those older blocks.
> > Please, come with suggestions and good ideas for a beginner...
> >
> > Today we tried a new antibody from Novocastra Laboratories (clone 5A5,
> > monoclonal) and I'm still having that pale brown color...
> >
> > The positive controls were pale too.
> >
> > Thanks in advance!

Tibor:

    Sounds like your DAB or peroxide are weak. Try fresh, as in buy a new
bottle, reagents. I have known DAB to work well one week and fail the next.
Also peroxide turns to water over time. In my lab, both of these reagents get
replaced every year. Cheap insurance.
    If that is not the problem, try adding nickel and cobalt to the DAB, gives
a blue-black reaction product that shows up very well. I can supply a recipe if
you like.

Geoff
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
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