Ab15249

We placed glass coverslips in a 6‐well culture plate and soaked them with 70% ethanol for 1 h. After washing the coverslips with PBS, we seeded about 3 × 10⁵ cells per well and cultured them overnight in culture media at 37 °C. The cells were then washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at RT. The slips were then rinsed with PBS, followed by permeabilization with 0.5% Triton X‐100 in PBS (PBS‐T; 30 min, RT). The cells were rinsed again with PBS, and the coverslips were moved to a paraffin‐covered plate that functioned as an incubation chamber. The cells were blocked with 1% BSA in 0.05% PBS‐T for 1 h at RT, using ≈250 µL of this blocking solution per coverslip. The blocking solution was aspirated, and the cells were incubated with 200–300 µL of CA125 (Abbiotec, 250 556), p53 (Leica Biosystems, CM5), Ki67 (Novus Bio, NB110‐89719), or WT1 (Abcam, ab15249) antibody solution diluted in the aforementioned blocking buffer. After incubation (1 h, RT), cells were triple‐washed with 0.05% PBS‐T in 5‐min intervals. Finally, cells were incubated with fluorophore‐conjugated secondary antibodies (1 h, RT) and then triple‐washed with 0.05% PBS‐T in 5‐min intervals. The coverslips were mounted onto slides using Fluoromount‐G, allowed to dry, sealed, and imaged using an EVOS FL Auto 2 microscope. We used ImageJ (version 10.2) to adjust for background fluorescence.

All antibodies were obtained from commercial sources. Staining for CD45 [FITC conjugated mouse monoclonal (B-A11), Abcam, ab27287] and CD71 [AlexaFluor647 conjugated mouse monoclonal (MEM75), Abcam, ab187777] was performed on ice for 30 min each. Additional WT-1 (1/50 dilution; rabbit anti-human, Abcam, ab15249) staining was performed on ice for 30 min after permeabilization with 0.1% Tween-PBS and blocking with 10% goat serum in FACS buffer. For flow cytometry, AlexaFluor405 goat anti-rabbit antibody (1/2,000 dilution; Abcam, ab175655) was used as a secondary antibody. For fluorescence histochemistry, FITC goat anti-rabbit antibody (1/100 dilution; Thermo Fischer, F-2765) was used as a secondary antibody to allow for Hoechst staining of the sections.

Rucaparib was kindly provided by Clovis Oncology. The following antibodies were used for WEHI PDX immunohistochemistry: p53 (M700101 1:100; Dako), Ki67 (M7240 1:50; Dako), Cytokeratin (Pan-CK; M3515 1:200; Dako), PAX8 (10336-1-AP 1:20000; Proteintech), and WT1 (ab15249; 1:800; Abcam). For DNA repair foci experiments, rabbit anti-human RAD51 (ab133534, Abcam) and rabbit anti-human γH2AX antibody (Phospho-Histone H2A.X Ser139; clone 20E3, #9718, Cell Signaling Technologies) were used. For BRCA1 immunoblotting, the mouse monoclonal antibody MS110 antibody was used (Calbiochem OP92).