This article describes a method for visualizing cholinergic neurons in the enteric nervous system of transgenic embryonic mice. The technique involves immunofluorescence staining and confocal microscopy to analyze the distribution of these neurons in the gastrointestinal tract.
Take a fixed, transgenic embryonic mouse gastrointestinal tract at the desired developmental stage.
The gastrointestinal tract's enteric nervous system includes enteric neurons and cholinergic neurons expressing choline acetyltransferase.
These cholinergic neurons also express a fluorescent protein driven by the choline acetyltransferase promoter.
Incubate the tract with a non-ionic detergent and proteins to permeabilize cells and prevent non-specific antibody binding.
Replace with primary antibodies. Incubate for the antibodies to bind their target proteins in neurons, respectively.
Remove the unbound antibodies and wash with buffer.
Incubate with fluorophore-conjugated secondary antibodies to specifically bind the primary antibodies bound to target proteins.
Remove the excess antibodies and wash with buffer.
Immerse the labeled tract in mounting media containing a nuclear dye to stain the nuclei. Now, mount a coverslip.
Using confocal microscopy, visualize and analyze the distribution of cholinergic neurons in the gastrointestinal tract.
Place the GI tracts into a 0.2-milliliter tube containing blocking solution, consisting of 1x PBS, 3% bovine serum albumin, 0.1 Triton X-100, and rock for 1 hour at room temperature. After blocking, aspirate the blocking solution with a fine-tipped Pasteur pipette and replace with the optimal dilution of primary antibodies diluted in blocking solution.
Incubate for either four hours at room temperature or overnight at 4 degrees Celsius on the rocking platform. Following the primary antibody incubation, aspirate the antibody solution and rinse the GI tracts in PBS three times for five minutes each, and then for one hour at room temperature with rocking.
After the last wash, replace the PBS with fluorophore-conjugated secondary antibodies diluted in blocking solution, and place the tubes on the rocking platform in the dark for four hours at room temperature, or overnight at 4 degrees Celsius.
After washing the GI tracts as before, place a few drops of fluorescence mounting media with DAPI onto a glass slide, then, immerse the GI tract into the mounting medium and add a cover glass. Finally, capture images of each fluorophore using a confocal microscope, then perform computer-aided image analysis using appropriate software.
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