An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons

Take three samples of hippocampal neuronal cultures on coverslips.

Introduce primary antibodies to label the surface-expressed glutamate receptors.

Add a conditioned medium to the second and third samples to induce endocytosis of the antibody-bound receptors.

On the third sample, add Fab fragments to block epitopes on the non-endocytosed antibodies.

Introduce an inhibitor to block receptor endocytosis while allowing receptor recycling to the surface.

In all samples, fix the cells and add a blocking solution to prevent non-specific labeling.&

Introduce fluorophore-tagged secondary antibodies to label the primary antibodies on the surface-bound receptors, including the recycled ones.

Permeabilize the cellular membranes.

Add the primary antibody again to the first sample to label the pre-existing endocytosed receptors.

Introduce a different fluorophore-tagged secondary antibody to all samples to label the endocytosed receptor-bound primary antibodies, distinguishing between endocytosed and surface-bound receptors.

Using a confocal microscope, quantify the surface-expressed, endocytosed, and recycled receptors in the different treatments.