An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons
Trascrizione
To begin this procedure, transfer the coverslips cell-side up to a paraffin film-covered tray to save reagents and facilitate manipulation. Save and maintain conditioned media at 37 degrees Celsius for incubation and washing steps.
Incubate the cells with primary antibodies diluted in conditioned media at room temperature for 15 minutes. After this, use a vacuum pipette to carefully aspirate off the antibody-containing media and wash the cells three times with conditioned media.
If studying surface versus intracellular receptor expression, fix the cells with paraformaldehyde after this step as outlined in the text protocol. Maintain the cells in conditioned media without antibodies and return them to the incubator at 37 degrees Celsius to allow for internalization. If studying internalization process, fix the cells with paraformaldehyde after this step, as outlined in the text protocol.
To block the epitopes on the primary antibody attached to the surface-expressed receptors that have not been internalized, incubate cells with unconjugated Fab anti-IgG antibody fragments diluted in conditioned media for 20 minutes at room temperature. After this, wash the cells three times with conditioned media. Incubate the washed wells with conditioned media containing 80 micromolar dynasore at 37 degrees Celsius to allow for recycling of internalized receptors.
After finishing the modifications on live cells, wash the cells once with PBS+. Add a solution of 4% paraformaldehyde and 4% sucrose in PBS to the cells, and incubate in a fume hood at room temperature for 7 to 8 minutes to fix the cells. Then, wash the cells three times with regular PBS. Add a solution of 10% normal goat serum in PBS to the cells, and incubate at room temperature for 30 minutes to block nonspecific binding sites.
Next, incubate the cells with fluorescently-tagged secondary antibody diluted in 3% NGS in PBS at room temperature for one hour to label primary antibody-labeled receptors. After this, wash the cells three times with PBS.
To begin labeling the intracellular receptors, add a solution of 0.25% Triton-X in PBS and incubate at room temperature for 5 to 10 minutes to permeabilize the cells. Then, block with 10% NGS at room temperature for 30 minutes. If studying surface versus total, incubate the cells with the same primary antibody used previously, diluted in 3% NGS in PBS at room temperature for one hour to label the intracellular receptors.
Next, wash the cells three times with PBS. Label with second fluorescently-tagged secondary antibody diluted in 3% NGS in PBS at room temperature for one hour. After this, wash the cells three times with PBS. First, gently place the coverslips, cell-side down, on 12 to 15 microliters of the appropriate mounting media to mount the cells. Then, image the cells on the appropriate confocal microscope, and analyze the cells as outlined in the text protocol.
