Immunostaining of Interneurons in a Rat Hippocampal Section

Take a fixed rat hippocampal section containing an interneuron filled with biocytin, a biotin-conjugated molecule. 

Next, add a solution containing blocking proteins and a non-ionic detergent to prevent non-specific binding and permeabilize neurons.

Incubate with a primary antibody targeting specific cannabinoid receptors on interneurons.

Wash with buffer to remove excess antibodies.

Then, add a streptavidin-conjugated fluorescent dye and a fluorophore-conjugated secondary antibody.

During incubation, streptavidin binds strongly to biocytin, allowing visualization of the biocytin-filled interneuron, while the secondary antibody targets receptor-bound primary antibodies on the interneurons.

Rinse with buffer, removing unbound molecules.

Now, mount the section using aqueous mounting media. Position a coverslip and seal it.

Using a fluorescence microscope, visualize the biocytin-filled interneuron and cannabinoid receptor expression, enabling analysis of the interneuron's morphology and receptor distribution.

Lock the tissues with 10% blocking serum for one hour at room temperature. Subsequently, incubate the sections in primary antibody CB1R at room temperature overnight, to identify cannabinoid receptor type one expression. Then, wash the brain sections three times for 10 minutes each in 0.1 molar PBS. Streptavidin and secondary antibody to first primary antibody, are both added to the well plate and kept overnight at four degrees Celsius.

Subsequently, incubate the sections in streptavidin red dye conjugate at four degrees Celsius overnight in the dark to reveal the biocytin. After that, wash the brain sections three times for 10 minutes each in 0.1 molar PBS. To protect the fluorescence and to facilitate restaining in the future, mount the sections in an aqueous-based mounting medium, and seal the edges of the coverslips with clear nail polish.