Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

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.