Solutions :

PBS buffer
PBS + 0.1% Tween (v/v)
DNA staining stock solution : 0.25 mg/ml DAPI in PBS, 50% glycerol (store at -20°C)
Mounting solution : add 2.4 g of Mowiol to 4.8ml of 100% glycerol, stir and add 6 ml H20. Stir at room temperature for several hours ; add 12 ml 0.2M Tris-HCl pH 8.5 and incubate at 50°C until dissolved. Add 0.45 g DABCO antifading reagent at 4°C (2.5% DABCO (w/v) final concentration). Aliquote and store at -20°C

Protocol description :

3T3 cells or HeLa cells are grown on coverslips. For PARP activity experiments, cells are exposed either to 1 mM hydrogen peroxide for 10 min or 1 mM MMS for 30 min, fixed with methanol / acetone (1/1, v/v) for 10 min at 4 °C and washed three times with phosphate-buffered saline supplemented with 0.1% Tween (v/v).

Primary antibodies :

Cells are incubated overnight at 4 °C with a monoclonal anti-poly(ADPribose) antibody (10H) (1:200 dilution) or with the polyclonal anti-poly(ADPribose) antibody (1:2.000).

Secondary antibodies :

After washing, the cells are incubated for 2 hr at room temperature (or for 4 hr at +4°C) with a 1:1.000 dilution of goat anti-mouse or goat-anti rabbit fluorophore-conjugated antibodies.

DNA staining :

The cells are washed 3 times in PBS. The last wash is done with a 1:20.000 dilution of the DNA staining stock solution. The coverslides are finally washed in water.

Mounting :

The coverslides are mounted using 25 µl of Mowiol containing DABCO antifading reagent. Care should be taken to avoid creating bubbles when depositing the coverslides on the mounting solution drop.

Immunofluorescence is evaluated using a Zeiss Axioplan microscope equipped with a DP50 Olympus CCD chilled camera or any equivalent device.