Anti mouse igg1 alexafluor 488

Cells were seeded onto coverslips in 6-well plates at a density of 3 × 10⁶ cells/well and grown overnight prior to incubation with BrdU (3 μg/ml) for 3 h at 37 °C. Cells were fixed with 4% paraformaldehyde for 60 min at room temperature and then washed five times with PBS for 20 min. For immunocytochemical staining, the cells were treated with 0.4% Triton X-100 containing 2 M HCl in PBS for 30 min. After washing with PBS, cells were blocked with 5% horse serum in PBS for 1 h at room temperature and subsequently incubated with mouse monoclonal anti-BrdU antibody (sc-sc-32323, Santa Cruz Biotechnology) at a dilution of 1:200 overnight 4 °C. On the next day, cells were washed in PBS three times and incubated with goat anti-mouse IgG1-Alexa Fluor 488 (A-21121, Invitrogen) at a dilution of 1:100 for 2 h at room temperature in the dark. Following washing three times with PBS each 5 min at room temperature, cells were stained with DAPI (1 μg/ml) for 2–3 min at room temperature to stain nuclei. Fluorescent images were acquired at 20× magnification using a Nikon TE300 digital inverted microscope, and the number of BrdU positive cells were determined by a laboratory member with no knowledge of sample identity using Image J software.

For immunofluorescent triple staining, the three most highly expressed markers for ductal and lobular ovarian metastases were chosen. In brief, FFPE sections of these ovarian metastases were deparaffinized as described above. Antigen retrieval was performed by placing the slides in EnVision Flex Target Retrieval Solution high pH (pH 9.0; Dako). Primary antibodies for ductal ovarian metastases: E-cadherin, EMA and Her2/neu. Primary antibodies for lobular ovarian metastases: EMA, Her2/neu and EpCAM. Secondary antibodies were all isotype-specific antibodies with Alexa Fluorochromes (LifeTechnologies, USA): anti-mouse IgG1-AlexaFluor488 (E-cadherin and EpCAM; green), anti-mouse IgG2a-AlexaFluor647 (EMA; red) and anti-rabbit-AlexaFluor546 (Her2/neu; orange). Sections were mounted with Vectashield containing DAPI (Vector Laboratories, USA). Primary invasive breast tumor samples that showed positive expression for all markers in previous experiments were used as a positive control. Tissue sections stained without application of primary antibodies were used as a negative control.

Antibodies targeting phospho-histone H2AX (γH2AX) (Millipore and Cell Signaling), TIF1β-pS824 (Kap1) (Cell Signaling), p53-pS15 (Cell Signaling), AID (Cell Signaling), Kin17 clone K58 (Santa Cruz), β actin (Sigma), GAPDH (Life Technologies), anti-mouse IgG1 Alexa Fluor 488 (Life Technologies), and anti-mouse Ig H+L Alexa Fluor 555 (Life Technologies) were used as specified by the manufacturers’ protocols for Western blot or Immunofluorescence analysis. The shRNA plasmids used in this study include pLKO.1-puro (The RNAi Consortium) and pLKO.005_TRC018-hygro (Gift from Jason Moffat). The CRISPR/Cas9 RNA expression plasmid px330 was use for CRISPR-mediated CSR assays (Zhang Lab, Addgene). DNA repair substrates included DR-GFP (Jasin Lab, Addgene), pIRES-TK-GFP (Gift from Takashi Kohno), and EJ2-GFP (Stark Lab, Addgene).