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Take a polymer-coated slide with mouse brain tissue sections containing dopamine neurons expressing tyrosine hydroxylase, a key enzyme for dopamine synthesis.
Outline the sections with a hydrophobic pen to ensure uniform staining.
Fix the tissue in an acetone-methanol solution to preserve tissue morphology.
Rinse with buffer containing detergent to permeabilize cell membranes.
Incubate with primary antibodies targeting tyrosine hydroxylase in dopamine neurons, then wash to remove unbound antibodies.
Add fluorescent dye-tagged secondary antibodies, nuclear dye, and RNase inhibitors.
During incubation, secondary antibodies bind to primary antibodies, nuclear dyes label the nucleus, and RNase inhibitors inactivate RNases.
Rinse with buffer. Then, dehydrate the tissue sections in increasing concentrations of ethanol.
Incubate in xylene to clear the tissue.
The processed tissue sections are now ready for laser capture microdissection, which allows precise visualization and dissection of target dopamine neurons.
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