High-Resolution Imaging of Neurotransmitter Receptors at Retinal Synapses

Take a mouse retinal section on an electron microscopy grid. The section contains retinal ganglion cells, or RGCs, synaptically linked to bipolar cells.

Presynaptic bipolar cells contain neurotransmitter vesicles arranged in a ribbon structure near the synaptic membrane.

Postsynaptic RGCs display clustered neurotransmitter receptors on their membrane. These cells are labeled with cholera toxin subunit B or CTB.

Wash with a buffer to maintain the pH.

Incubate with a blocking buffer to mask non-specific binding sites.

Incubate with primary antibodies targeting the neurotransmitter receptors and CTB.

Wash with a buffer to remove unbound antibodies and maintain the pH.

Incubate with secondary antibodies conjugated to gold nanoparticles of different sizes, which bind to the primary antibodies, labeling the target molecules.

Remove unbound antibodies, then air dry the sample.

Apply heavy metals that bind macromolecules, enhancing the contrast.

Using electron microscopy, identify the RGCs using the labeled CTBs and locate the labeled neurotransmitter receptors.

In this step, cut 70-nanometer thick, ultra-thin sections with an ultramicrotome and collect them on the Formvar-carbon-coated nickel g