Cells are grown on coverslips (round or rectangular) processed for immunocytochemistry and mounted on slides for observation from the coverslip side. Antibody incubations need be in small volumes to save precious antibodies and wash volumes have to be generous to ensure proper wash of reagents. To incubate primary antibodies on coverslips, our laboratory uses a home-made incubation tray. We mounted 5/8" rubber "O"-rings into a 6x7.5" plastic tray in a 5 across by 3 down array using super glue.
1. Remove coverslips from culture dishes into a Coors holder (Thomas Scientific, part # 8542E40) immersed in PBS.
2. Transfer holder to 4% Paraformaldehyde and fix for 10min at RT.
3. Wash samples in PBS 3x10 min (pH 7.4).
4. Permeabilize samples in ice-cold methanol for 3 min at -20°C.
5. Wash samples in PBS for 3x10min.
6. Transfer coverslip from PBS wash on top of a tray's ring, cell side up. Add 500 µl of antibody dilution on each coverslip. Antibodies should be diluted into 10% serum of the host from the secondary antibody. Humidity is maintained by placing a moist strip of cut sponge at one end of the tray and cover the tray. Shaking is not required for primary antibodies. Incubate overnight at 4°C. Return coverslips to the Coors holder for washing and incubation with secondary antibodies en masse.
7. Wash samples in PBS for 3x10min.
8. Incubate in appropriate secondary antibodies for 1hr. at RT. 1:200 in normal serum from the host of the secondary antibody.
9. Wash samples in PBS for 3x10min.
10. Dab coverslip one at a time on kimwipes to remove excess PBS. DO NOT LET CELLS ON COVERSLIP DRY!
11. Apply 20µl MOWIOL mounting media to a clean dry slide
12. Touch the edge of the coverslip to MOWIOL, then lower slowly.
13. Avoid air bubbles at all costs!
14. Store flat and dark overnight. Store long term in refrigerator in the dark.
