When you activate a structure like the RMTg, it exerts GABAergic effects on the VTA, which is dopaminergic. After slicing the tissue, is it possible to get a good stain of dopamine levels in various parts of the brain using dopamine antibody? Or will you just have too much background?
For further clarification of this question, I would compare it to determining neuronal c-Fos expression after i.p. amphetamine administration.
IHC is the best way to show specific neurons. Bu it is difficult to get quantitative results. The first way: it may be usefull to perform protein analysis and immunohistochemistry together. In this way, dissection of the target nucleus is going to be a problem for protein analysis. Second way: use serial sections for immunostaining (The first section for dopamine, the second for c-Fos and so on), and stereological methods for evaluation.
One classical method for the examination of dopamine using IHC like applications is the use of formaldehyde. Catecholamines produce a characteristic fluorescence known as formaldehyde-induced fluorescence. You could potentially take advantage of this in combination with other fluorescently labeled antibodies (just make sure that they excitation emission spectra don't overlap. There are a lot of papers that describe FIF methods so it really depends on your experimental setup. Good luck!
Thank you all very much! This is all helpful information, and gives me a good idea of the direction I should be heading.
Dear Deborah - I am thinking of trying IHC to detect intracellular dopamine diffusion in a cellular network in brain slices. Have you tried it in the end? Would you have an antibody to advise? Many thanks for your help!
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