Hi Johanna:

    Your proceedure sounds fine to me. I have never used the antibody in 
question but getting good results with TH should be VERY easy. I use a 
polyclonal TH from Pel-Freez in Rogers, AK, works great on both mouse 
and rat brains. Assuming the brains were not overfixed (24 hours could 
be too long)  and the antibody is still good I would make sure all of my 
solutions were properly labeled. Also, is your detection system OK? You 
could have great binding but if the detection system is not working 
properly you will never know it.

Geoff

Jackson, Johanna wrote:

>Dear All,
> 
>I am trying to stain dopaminergic neurons in the striatum of the adult rat brain which has been perfused with 4% PFA, washed and then cryoprotected in 20% sucrose, before being sectioned (10um) on a cryostat. I pretreat the sections with 0.1% Triton-X and block using serum.   I am using the monoclonal anti-tyrosine hydroxylase antibody from Sigma (T1299).  Sigma recommend a working concentration of 1:2000 however I have had no luck with this staining.  I just a weak staining all over the brain with no specificity in the striatum.  
> 
>Does anyone have a protocol using this particular antibody? I have seen protocols with anti-TH from other companies which follow a similar method to what I have described above, however they do not seem to work for the Sigma antibody.  
> 
>Any help would be greatly appreciated!
> 
>Thanks!
> 
>Jo
> 
> 
>Stem Cell Imaging
>MRC Clinical Sciences Centre
>Imperial College London
>Hammersmith Hospital Campus
>Du Cane Road
>London
>W12 0NN
>0208 3833796
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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@umdnj.edu
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