Re: [Histonet] Problems with c-fos immediate early gene labelling

From:Geoff McAuliffe

Hi Ron: 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.


>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
>Histonet mailing list

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

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