Immunocytochemistry/Immunofluorescence Protocol for Separase Antibody (NB100-330)

Immunofluorescence Procedure Cell Preparation
1. HeLa cells are grown on coverslips and enriched in a fraction of mitotic cells by double thymidine block/release protocol. 2. Briefly, cells are allowed to attach to the glass coverslips for 16 hours. 3. Thymidine is added to the culturing medium at a final concentration of 2 mM for 18 hours. 4. Cells are washed 2 times with PBS. 5. Fresh medium without thymidine is added. 6. Cells are incubated in the thymidine-free medium for 8 hours. 7. Thymidine is added again to a final 2 mM concentration for 18 hours again. 8. After the second thymidine incubation, the cells are washed 2 times with PBS. 9. Fresh thymidine-free medium is added again. 10 .Starting at 8 hours after the second thymidine removal, cells are analyzed by light microscopy and once population of mitotic cells are about 50% (usually around 9-10 hours after the second release) the cell staining procedure begins.
Cell Staining 1. Cells are washed 2 times with Dulbecco's PBS with Ca and Mg (D-PBS) and permeabilized prior to fixation by incubation with 0.05% Triton X-100 for 1 min. 2. Permeabilizing solution is aspirated and 4% paraformaldehyde is added for 30-45 min. 3. Paraformaldehyde is removed by washing the coverslips 3 times with D-PBS. 4. Cells are blocked by SuperBlock reagent (Pierce) in PBS supplemented with 0.5% Triton X-100 for 30 min. 5. The coverslips are washed 3 times with 0.05% Tween-20 in PBS (PBS-T). 6. Staining to detect separase is carried out at a dilution of 1:1,000 of mouse monoclonal anti-separase (NB 100-330) prepared in PBS-T and incubated with the cells on coverslips for 1 hour at room temperature (RT). 7. Coverslips are washed 3 x 5 min. washes with PBS-T. 8. Incubate cells with an anti-mouse Cy3 conjugated fluorochrome-labeled secondary. The secondary antibody is diluted in PBS-T and incubated for 20 min. 9. Coverslips are incubated with DAPI at a final concentration of 10 ng/ml in PBS-T for 5 min. 10.Cells are washed 5 x 5 min with PBS-T. 11.Prior to mounting, the coverslips are washed once with PBS without detergents. 12. Micrographs are taken by a fluorescent microscope.