Dapi mountant

Hepatocytes were cultured on sterile collagen-coated coverslips, then fixed in
2% paraformaldehyde for 10 min and then blocked cells for
45 min with 0.2% Fish skin Gelatine, 0.5% BSA
and 0.5% Triton X-100 in PBS. Slides were then incubated with the
rabbit polyclonal anti-53BP1
4 μg ml⁻¹ (NB100-904
Novus Biologicals) overnight at 4 °C. The next day, the cells
were washed and incubated with Alexa Fluor 594 secondary antibody (Invitrogen)
for 60 min before mounting with DAPI mountant (Vector).

Plates were thawed, rehydrated and washed 4 × 2 min with 300 µL well⁻¹ PBST (0.01 M PBS, 0.05% Tween-20). The cells were treated with 250 µL well⁻¹ 5% skimmed milk powder (SMP) in PBST for 1 h at 22 °C. After a further wash with PBST the two affinity-purified MAbs (20 µg mL⁻¹) were added to the plate (100 μL) and incubated for 1 h at 22 °C. The plates were washed again and 100 µL goat anti-mouse IgG fluorescein isothiocyanate (FITC)-conjugated MAbs (Sigma-Aldrich, USA), diluted 1/100 in PBS, were added to the wells and incubated for 1 h at 22 °C. The plates were washed a final time and kept in the dark at 4 °C prior to reading the fluorescence in a Synergy HT spectrophotometer (Fisher Scientific, Leicestershire, UK). The Gen 51.10 program was used for data acquisition at a fluorescence detection sensitivity setting of 120, with filter settings applied at wavelengths of 485/20 excitation and 528/20 emission.
Once the FITC fluorescence had been measured, 50 µL DAPI mountant (Vectashield, Vector, UK), diluted 1:10 in PBS, was added to all wells and incubated at 22 °C for 10 min. Excess DAPI was removed from the wells by 4 × 2 min washes with PBST. The plates were read a second time using the same program and sensitivity settings, but with filter sets of 360/40 excitation and 460/40 emission.