Immunohistochemistry of Mouse Brain Tissue Sections for Targeting Dopamine Neurons

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.

To carry out immunohistochemistry, use a hydrophobic pen to outline tissue and allow them to dry. Then, in a 1 to 1 acetone methanol solution, fix the tissue at negative 20 degrees Celsius for 10 minutes. After removing the tissue from the freezer, cover the sections with 100 to 200 microliters of tyrosine hydroxylase antibody in PBS and 1% triton for 10 minutes.

Then, use PBS with 1% triton to rinse the slides before covering the sections with 100 to 200 microliters of goat antirabbit IgG, labeled with Alexa Fluor 488. Incubate for five minutes, and then use PBS to rinse the slides twice. Next, dehydrate the samples in a graded series of RNase free ethanol for 30 seconds each, as demonstrated earlier in this video. Incubate in xylene, for one minute and a second wash, for five minutes. Remove the slides immediately prior to use for LCM and allow them to air dry.