Alexa 647 conjugated donkey anti rabbit

Immunofluorescence procedures were performed as previously described28 (link). Cell proliferation was detected using a primary polyclonal antibody against PCNA (Abcam, ab2426). Immunolabeled proteins were visualized using Alexa 647-conjugated donkey anti-rabbit (Invitrogen, Grand Island, NY, USA), Cy3-conjugated donkey anti-chicken (Jackson ImmunoResearch laboratories Inc., West Grove, PA, USA), and FITC-conjugated donkey anti-goat antibodies (Jackson ImmunoResearch Lab), as appropriate. For YAP1 immunofluorescence staining, anti-YAP1 antibody was used (Cell Signaling, cat 4912 and Anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor^® 594 Conjugate) cat 8889). Tissues were mounted in Vectashield mounting medium (Vector Laboratories, CA, USA). Images were acquired using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany) and LSM 510 version 2.02 software.

For BrdU incorporation analysis, MEFs were pulse-labeled for 1 h with 100 μM BrdU (BD Pharmingen, 550891) before collection. Collected cells were fixed in –20 °C cold 70% ethanol and washed in blocking buffer (PBS-bovine serum albumin (BSA) 1%; BSA Sigma-Aldrich, A7906). For non-BrdU analysis, cells were permeabilized with PBS-1% BSA-0.5% Triton X-100, whereas, for BrdU analysis, cells were treated with 2 n HCl/0.5% Triton X-100 and neutralized with 0.1 m sodium tetraborate, pH8.5. Cell staining were performed using anti-BrdU (1:500; BD Pharmingen, 555627, clone 3D4), pS10-histone H3 (1:100; Cell Signaling Technology [CST] #9701) or pS139-histone H2AX (1:500; CST #9718) followed by their incubation with secondary antibody Alexa 647-conjugated goat anti-mouse (1:400; Invitrogen, A21235) or Alexa 647-conjugated donkey anti-rabbit (1:400; Invitrogen, A31573). Before analysis, cells were resuspended in PBS supplemented with 2.5 μg/ml propidium iodide (Merck, 537059) and 20 μg/ml RNase A (Sigma-Aldrich, R6513). Analysis of cells and BrdU incorporation was acquired on BD FACSCalibur or BD LSRII flow cytometers (BD Bioscience) and further analyzed using FlowJo 8.8.7 software.