Hi everybody,
I try to use tyrosine hydroxylase antibody (Millipore-Sigma, #MAB318) to stain dopaminergic neurons in zebrafish at 5dpf. The problem is : I have a good staining of zebrafish treat with MPTP but not so good in my control. I suppose that the antibody does not cross well the hematoencephalic brain barrier. Actually I fix o/n at 4C with 4%PFA, permeabilize the fish 15 min in cold acetone at -20C, and make every wash/blockage with 3-4% triton. I try a lot of protocol ! so if anybody has been the same problem and has find a solution, that will help me a lot !
Thank you so much !
try aiding permabilisation with MeOH -20C, O/N.
have you try fixing with 2% TCA
Hi Mathilde,
If the staining has worked in treated but not control, it could be an experimental error. I have not used this specific antibody. However I have used Millipore AB152 and Immunostar 22941, both have worked very well in 3 and 5dpf zebrafish larvae (both in wt and MPTP treated ones).
As you have not described the complete protocol, I am only going to comment on the steps that you have mentioned :
Permeabilization: I use proteinase k ( 10 µg /ml in PBS) 20 min- 30 min at RT for permeabilization. I have used acetone, but proteinase k gave me better results with TH Antibody.
Washing: I use 0.5% Triton in PBS for washing.
I am attaching my protocol for your reference
All the best
Smijin
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