Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine 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, an