Immunostaining of Neurons in Mouse Brain Slices Following Fluorescence In Situ Hybridization

Take fixed and permeabilized mouse brain slices containing neurons, including cholinergic neurons expressing choline acetyltransferase.

The slices are subjected to fluorescent in situ hybridization to detect target mRNAs localized to cholinergic axons, resulting in amplified signals using fluorescent probes, which are detected with fluorescence microscopy.

Add a blocking solution containing proteins and a quenching agent. The proteins prevent non-specific binding, and the quenching agent neutralizes residual fixatives.

Incubate with primary antibodies that target neuronal choline acetyltransferase.

Wash with buffer to remove unbound antibodies.

Incubate with fluorophore-conjugated secondary antibodies that bind to the choline acetyltransferase-bound primary antibodies.

Wash with buffer to remove excess antibodies, then rinse in distilled water to remove buffer residues.

Mount the slices with mounting media containing a nuclear stain to label the nuclei.

Using fluorescence microscopy, image the slices to visualize the target mRNA signals and cholinergic axons stained with specific antibodies.