Wikipedia says: Immunohistochemistry (IHC) refers to the process of selectively imaging antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
We use this in our work because sometimes we want to know if a given protein can be found in a given brain area or cell type. For example, perhaps we want to know if a given brain area was highly active. We could image for cFos in that area. Other times we might want to know if two proteins were in the same cells. For example, if we infect the brain with a virus that should only express green fluorescent protein (GFP) in acetylcholine producing cells, we might want to stain for the enzyme that constructs acetylcholine (Choline Acetyltransferase, AKA ChAT).Â
The process uses a bit of chemistry and a bit of magic (i.e., obsessive compulsive following of a protocol).
In most protocols you first wash your brain slices in a mild detergent - this loosely breaks holes into the lipid membrane of the cells, thereby allowing the anti-bodies into your cells so as to find the target protein you are looking for. Then, you bath the slices in a serum from the same animal from which your fluorescent anti-bodies will come from - this helps to reduce background staining because those anti-bodies are less likely to stick to the serum of the animal that made them. Finally, you start with the anti-bodies. There are two rounds of anti-body bathing, the first is just to find the target proteins, the second is to add the fluorescent tag. Thus, you bath the tissue in the first anti-body for a while (usually overnight) then wash the slices very carefully. The washing is important because the next set of anti-bodies will not be looking for your target protein but rather the first anti-body you used. Washing will get rid of any anti-bodies that are not bound to your target protein and thus help get rid of background staining (improving your signal quality!). Now you bath the slices in the fluorescent anti-bodies. Finally, wash the slices very carefully again. Just like before, this is important for getting rid of background staining. And you're done! You can mount your slices to slides, coverslip, and check 'em out under a microscope!
EYFP or GFP: A method for amplifying virally transduced EYFP or GFP reporters
ChAT: A method for visualizing the expression of Choline Acetyltransferase (ChAT), the enzyme responsible for the production of acetylcholine.