This article outlines a detailed immunohistochemical protocol for visualizing choline acetyltransferase in mouse brain sections. The method includes steps for tissue preparation, antibody application, and visualization techniques.
Take a fixed mouse brain section containing the nucleus basalis of Meynert, rich in nerve fibers expressing the enzyme choline acetyltransferase.
The section is pretreated to deactivate endogenous peroxidase.
Add a detergent to permeabilize cellular membranes, followed by a blocking solution to prevent non-specific antibody binding.
Replace the solution with primary antibodies that bind to choline acetyltransferase.
Wash with buffer to remove unbound antibodies. Incubate with biotin-tagged secondary antibodies, which bind to the primary antibodies.
Remove unbound antibodies and add avidin-biotin-peroxidase complexes, which bind to the secondary antibodies.
Wash to remove excess complexes.
Add a chromogenic substrate that reacts with peroxidase, forming a colored precipitate.
Rinse with distilled water to remove excess precipitate.
Transfer the section onto a gelatinized slide.
Next, dehydrate the section using increasing ethanol concentrations, followed by xylene.
Mount the section and observe under a microscope to visualize the stained nerve fibers.
For immunohistochemical labeling of the tissue sections, after blocking any nonspecific binding with 10% normal bovine serum in 0.1% Triton X-100, in tris-buffered saline, label the sections with the primary antibody of interest, for 48 hours at four degrees Celsius.
At the end of the incubation, wash the sections three times, for three minutes, in fresh tris-buffered saline per wash. Followed by incubation with an appropriate biotinylated secondary antibody, for one hour at room temperature.
At the end of the incubation, wash the sections three times in fresh tris-buffered saline, as demonstrated. Followed by staining with avidin-biotin-peroxidase complex and DAB peroxidase substrate solution, according to the manufacturer's protocols.
After washing, mount all of the sections from one brain tissue sample onto one gelatinized slide. Allow the samples to air dry. To dehydrate the sections, immerse the samples two times, for five minutes per dilution, in sequential ascending ethanol solutions, as indicated, followed by clearing with two 10-minute xylene washes. Then, use an appropriate mounting medium to place coverslips onto the slides.
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