[Histonet] tyrosine hydroxylase staining??

[Jackson, Johanna]

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