Fixed specimens at an optimal concentration were placed onto a 300 mesh
carbon/formvar coated grids and allowed to absorb to the formvar for a minimum
of 1 minute. For immunogold staining the grids were placed into a blocking
buffer for a block/permeabilization step for 1 hour. Without rinsing, the grids
were immediately placed into the primary antibody at the appropriate dilution
overnight at 4°C (monoclonal anti-CD9 1:10, Abcam). As controls, some
grids were not exposed to the primary antibody. The next day, all of the grids
were rinsed with PBS then floated on drops of the appropriate secondary antibody
attached with 10nm gold particles (AURION, Hatfield, PA) for 2 hours at room
temperature. Grids were rinsed with PBS and were placed in 2.5%
Glutaraldehyde in 0.1M phosphate buffer for 15 minutes. After rinsing in PBS and
distilled water the grids were allowed to dry and stained for contrast using
uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmission
electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD
Camera (Advanced Microscopy Techniques, Danvers, MA).
carbon/formvar coated grids and allowed to absorb to the formvar for a minimum
of 1 minute. For immunogold staining the grids were placed into a blocking
buffer for a block/permeabilization step for 1 hour. Without rinsing, the grids
were immediately placed into the primary antibody at the appropriate dilution
overnight at 4°C (monoclonal anti-CD9 1:10, Abcam). As controls, some
grids were not exposed to the primary antibody. The next day, all of the grids
were rinsed with PBS then floated on drops of the appropriate secondary antibody
attached with 10nm gold particles (AURION, Hatfield, PA) for 2 hours at room
temperature. Grids were rinsed with PBS and were placed in 2.5%
Glutaraldehyde in 0.1M phosphate buffer for 15 minutes. After rinsing in PBS and
distilled water the grids were allowed to dry and stained for contrast using
uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmission
electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD
Camera (Advanced Microscopy Techniques, Danvers, MA).