Imaging cell cultures

A project by students who cultured the cells in a laboratory and sorted them using the Cytometry Facility at University Park.

Protocol

1. Seed RAW264.7 cells onto Thermanox coverslips seated in a 24 well plate and grow to confuency. Incubate the cells with 40nm colloidal gold overnight.
2. Remove the media and rinse with HBSS
3. Fix with 1.5% glutaraldehyde, 2.5% paraformaldehyde in 0.1M sodium phosphate buffer (pH=7.4) for 2 hrs at 4 C
4. Rinse with 0.1M sodium phosphate buffer (3x5min)
5. Postfix with 1% osmium tetroxide in 0.1M sodium phosphate buffer for 1 hr.
6. Rinse with 0.1M sodium phosphate buffer (3x5min) Note: transfer the coverslips from the 24 well plate to the critical point dryer specimen holder for 13 mm coverslips during the last buffer wash
7. Dehydrate through a gradient series of ethanol (25%, 50%, 70%, 85%, 95% and 100% (x3) for 5 minutes at room Temperature
8. Critical Point Dry the samples with bone-dry liquid carbon dioxide (4 x 3 minute exchanges + 8 minute vent)
9. Mount the coverslips onto an aluminum stub with a double sticky tab and colloidal silver
10. Evaporate carbon onto the surface (10-2 mbars, 3 x 1 sec flashes) using the Carbon threads and the evap mode on the SCD050 sputter-coater