Re: [Histonet] tyrosine hydroxylase staining??
Just curious...when you say you get weak, diffuse staining all over the brain, is that in sections that should contain dopaminergic cell bodies (like in the midbrain, hypothalamus, etc.) or in areas where you would mostly see diffuse fiber staining (like in the striatum)? Because I've started staining for TH in rodent brain and the staining pattern in the striatum is diffuse (i.e. the whole striatum is darker than the surrounding cortex, but nothing like cell bodies...because it's mostly terminal projections- correct me if I'm wrong, but I wouldn't expect to see many/any cell bodies labeled with TH in the striatum), whereas it clearly picks up cell bodies in regions that contain dopamine producing neurons.
So if you were only staining rostral forebrain sections (like the striatum), I don't know if you should expect to see cell bodies, just diffuse fibers. If you're not already doing it, maybe you should include more caudal sections in your IHC (sections containing the arcuate nucleus or ventral tegmental area, etc. would be great)...that way you'd know if you're detecting cells in those areas and the weak staining in striatum could be fibers that just need amplification? Sorry if I'm misinterpreting your post about the weak staining...but it would be an easy thing to overlook if you didn't have sections with cell bodies in them...
Finally, I've only used a sheep anti-TH IgG from Novus Biological and it worked beautifully on the first time in a non-traditional rodent species (1:1,000 with fluorescence, so I imagine even more dilute concentrations should work with enzyme-mediated chromagen detection)...so I agree with Geoff McAuliffe that getting good results with TH should be easy...
If you're interested I can email you my protocol, it's not too different from what you described....but what detection system are you using? Fluorescence, DAB, other? Because you didn't mention any pretreatments like HRP inactivation or quenching unreacted aldehydes...maybe if you give more particulars to your methods, others could provide more tips?
Good luck with the trouble shooting,
Geoff McAuliffe wrote: 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.
Jackson, Johanna wrote:
>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!
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