This article describes a detailed protocol for examining retinal ganglion cells (RGCs) using electron microscopy. The method involves labeling cells with cholera toxin subunit B and antibodies to visualize neurotransmitter receptors.
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 grids. Wash the grids in distilled water once, followed by three washes of TBS. Then, incubate the grids in 20 microliters of 5% BSA in TBS for 30 minutes, and then in 20 microliters of a mixture containing goat anti-CTB and an antibody to one NMDAR subunit overnight at room temperature.
After that, wash the grids three times with 20 microliters of TBS each time at pH 7.6. Subsequently, wash the grids once with TBS at pH 8.2 for five minutes. Incubate the grids for two hours with 20 microliters of a mixture containing donkey anti-rabbit IgG, coupled to 10 nanometer gold particles, and donkey anti-goat IgG coupled to 18 nanometer gold particles.
After two hours, wash the grids in 20 microliters of TBS at pH 7.6 three times, followed by a wash in the ultrapure water. And then air dry the grids. Subsequently, counterstain the grids with 5% uranyl acetate in distilled water for eight minutes, followed by 0.3% lead citrate in distilled water for five minutes in the dark.
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