This article describes a method for detecting locus coeruleus regions in the mouse brainstem, which contain norepinephrine-producing neurons. The process involves the use of specific antibodies and fluorescence microscopy to visualize these neurons.
The locus coeruleus regions, located bilaterally in the mouse brainstem, contain norepinephrine-producing neurons and regulate various regions across the brain.
To detect these locus coeruleus regions, begin with a glass slide carrying the fixed, cryopreserved mouse brainstem sections.
Wash the slide with a buffer to remove fixative traces.
Add a buffer containing detergent and incubate to permeabilize the membranes, then wash.
Introduce primary antibodies specific to dopamine β-hydroxylase enzyme, which converts dopamine to norepinephrine, a neurotransmitter.
Incubate to allow the antibodies to interact with dopamine β-hydroxylase.
Wash to remove unbound antibodies.
Then, add green fluorophore-coupled secondary antibodies and incubate in the dark.
The secondary antibodies bind to the primary antibodies and then wash.
Add mounting medium to the brain sections, then cover and dry.
Observe the slides under a fluorescence microscope.
The green fluorescence representing the labeled norepinephrine-producing neurons in the brain sections indicates the presence of locus coeruleus regions.
On the first day, wash the brain slices in the 24-well plate 3 times for 5 minutes each in PBS. Then, add 0.5% PBS with detergent, and permeabilize them at 4 degrees Celsius for 24 hours.
On the following day, wash the slices three times for 5 minutes each with 0.5% PBSD. Then, add the primary antibody at a dilution of 1 to 500 in 0.5% PBSD, and incubate at 4 degrees Celsius for 18 hours.
On the 3rd day, wash the slices 3 times for 10 minutes each with 0.5% PBSD. Then, add the secondary antibody at a dilution of 1 to 1,000 in 0.5% PBSD. Wrap the plate with aluminum foil and incubate at 4 degrees Celsius for 16 hours.
After incubation, wash the slices 3 times for 5 minutes each with 0.5% PBSD, and then for 5 minutes in PBS. Cover the sections using hard-set mounting medium without DAPI. Cover with a glass cover and dry for 30 minutes at room temperature. Then, use a confocal microscope with settings to detect signal from appropriate fluorescence wavelength, to image brain slices.
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