[Histonet] Problems with c-fos immediate early gene labelling
Geoff McAuliffe
mcauliff <@t> umdnj.edu
Tue Mar 23 12:24:38 CST 2004
Hi Ron:
komo <@t> bu.edu wrote:
>I'm hoping I could get some helpful advice on what might cause my c-fos
>IHC labelling to work well in one lab, but not my own. I am running the
>protocol on rat brains using a hydrogen peroxide/methanol step for
>endogenous peroxidase blocking, a normal goat serum blocking step, a
>c-fos Ab-5 primary from oncogene, a goat-anti rabbit biotinylated
>secondary, a streptavidin horeradish peroxidase, and a nickle enhanced
>DAB reaction.
>
>Unfortunately, although I have all of the exact same antibodies, chemicals, and concentrations as the lab from which the protocol was developed, the label is weak and not very specific when I run it at my home lab. In fact, I even took brain slices from the same rats that I was running at my lab and ran them at the lab where the protocol was developed, and found that the c-fos label was beautiful with a low background.
>
When you say you are using the "exact same antibodies, chemicals,
and ..... " do you mean the same bottle of reagent? You are carrying
the bottle or vial of reagent from one lab to another? If not, look at
the batch of antibody, your peroxide, your DAB, etc. If you are storing
antibody in a freezer at home it is being subjected to freeze-thaw
cycles (unless you have a very old freezer that does not defrost itself
on a timer). If your bottle of peroxide is more than a few months old it
may be in the process of turning into water, peroxide does that. I have
had DAB work one day and fail the next.
Since you have found that the brain slices stain well in the "other
lab", the problem must lie with your home lab. Since bleach reacts with
DAB I would avoid using staining dishes treated with bleach.
Geoff
>Does anyone has any suggestions as to why this might be? One person
>at the other lab suggested that possibly the wells that we run the tissue in
>were not being cleaned properly (we soak them in bleach while the other
>lab uses just pex). Could this have any effect?
>
>Thanks for any advice you can provide!!
>
>Rob Komorowski
>
>
>
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>
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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 <@t> umdnj.edu
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