This article describes a protocol for visualizing neuronal membrane proteins in mouse brain tissue using transmission electron microscopy (TEM). The process involves freeze-fracturing the tissue, antibody labeling, and imaging to identify specific proteins.
Take a freeze-fractured specimen of mouse brain tissue in a buffer.
The specimen is coated with platinum to enhance surface details and with carbon for structural stability.
Transfer the specimen to a vial with a digestion buffer containing detergent.
Incubate to facilitate tissue breakdown, leaving intact the membrane and proteins embedded in the coating.
Wash with buffer to remove debris.
Add a blocking solution to prevent non-specific antibody binding.
Introduce primary antibodies specific to target neuronal membrane protein.
Wash with buffer to remove unbound antibodies.
Incubate with gold-conjugated secondary antibodies that bind to primary antibodies.
Wash with buffer to remove excess antibodies.
Rinse with water to prevent artifacts from salt residues during microscopy.
Mount the specimen on a transmission electron microscope or TEM grid.
Under the TEM, visualize the target neuronal membrane protein identified by gold particles, which appear as black dots.
For SDS digestion, transfer a replica to a 4 milliliter glass vial filled with 1 milliliter of SDS-digestion buffer. Allow it to digest for 18 hours at 80 degrees Celsius with shaking. For immunolabeling, wash the replica for 10 minutes in fresh SDS-digestion buffer. Then, incubate it with primary and secondary antibodies diluted in 2% BSA TBS in a humid chamber at 15 degrees Celsius for 24 to 72 hours.
After that, mount the replica on a formvar-coated, 100-line parallel bar grid. Image the replica with a transmission electron microscope at 80 or 100 kilovolts. Then acquire the digital images through a CCD camera.
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