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.

Cover each slice with 100 to 200 microliters of blocking solution, containing three milligrams per milliliter BSA, 100 millimolar glycine, and 0.25% Triton X-100 in PBS. Cover each slide with parafilm, and incubate for 30 minutes at room temperature.

Following the incubation, at 100 to 200 microliters of antibody diluted in blocking solution to the slides, an anti-choline acetyltransferase antibody is used here. After covering, the slides with parafilm, incubate for two days at four degrees Celsius. Make sure that brain slices don't dry out. If needed, reapply anti-chAT antibody solution to brain slices 24 hours after incubation.

After washing the slides three times in 1X PBS for five minutes at room temperature, add 100 to 200 microliters of the appropriate Alexa conjugated secondary antibody to the slides. Cover with parafilm and incubate for one hour at room temperature while protected from light.

After the hour has elapsed, wash the slides three times in 1X PBS for five minutes each as before, and then wash once with distilled water. Mount the slides with mounting medium containing DAPI. And then visualize the brain slices under the fluorescence microscope.