53BP1 foci

1. Culture cells (300,000-400,000) on glass coverslip (6 well dish) for 24 hours prior to treatment with aphidicolin.
2. Add 0.2 uM aphidicolin to treated wells and incubated at 37C for 24 hours.

Fixation and Permeabilization

1. Aspirate off DMEM and rinse once in PBS. Aspirate off PBS.
2. Thaw tube of 3% paraformaldehyde/2% sucrose fixative in water bath. Add 2ml paraformaldehyde/sucrose solution to each well of cells and incubated for 10 min at room temperature.
3. Rinse 2 times with PBS, aspirated off. Add 0.5ml of cold Triton X-100 solution to each well and incubate 5 minutes on ice.
4. Rinse well 4-5 times in PBS.
5. Blocked with 5% BSA in PBS for 15 minutes at room temperature.

Immunostaining

1. Spin antibodies down for 5 minutes at 13,000 rpm to remove debris (MAB 33802 mouse anti-53BP1, Sc-571 rabbit anti-Cyclin A).
2. Place parafilm on top of lid of plate and made dilution of MAB33802 at 1:500 and Sc-571 at 1:200. Diluted antibodies in 3% BSA.
3. If using small coverslips put 50ul (100ul for large coverslips) of antibody dilution onto parafilm and place coverslip onto the antibody solution with the cell side facing down.
4. Incubated for 1 hour at 37C in humidity chamber to prevent drying out.
5. Rinse twice in PBS for 2 minutes each wash.
6. Repeat steps 6-9 with secondary antibody. Used Alexaflour 594 goat anti-mouse (red) at 1:500 and Alexaflour 488 goat anti-rabbit (green) and incubated for 30 minutes at 37C in humidity chamber.
7. Wash with PBS 3 times.
8. Label slides and place 5ul of Prolong Gold+DAPI mounting solution onto slide.
9. Pull up coverslip out of well and dry the back with vacuum (non-cell side) and place cell side down onto slide containing Prolong.
10. Gently push down with syringe tip to remove bubbles and then press gently with whatman paper.
11. Store in slide holder at room temperature and let sit overnight.