Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

Take a chemically fixed mouse brain slice embedded in a tissue-embedding medium.

Wash the slice with buffer to remove the medium.

Add a solution that permeabilizes the membranes and blocks non-specific binding sites.

Remove the solution.

To visualize neuronal synaptic proteins, incubate the slice with primary antibodies.

These antibodies target a presynaptic protein expressed in the synapses of specific brain regions and a ubiquitously expressed synaptic protein as a reference marker.

Wash with buffer to remove unbound antibodies.

Introduce fluorophore-labeled secondary antibodies that target the primary antibodies.

Wash with buffer to remove unbound secondary antibodies.

Counterstain the nuclei with a DNA-binding dye.

Wash with buffer and mount the slice using a suitable mounting medium.

Visualize the slice under a confocal microscope.

Calculate the mean fluorescence intensity ratios of the target protein and the reference marker to analyze the target protein's distribution across different brain regions.