Alexa fluor 488 chicken anti rabbit igg h l

Cells were fixed in 4% Paraformaldehyde (PFA, Electron Microscopy Sciences) for 10 min at RT followed by 10 min of permeabilization using the following permeabilization buffer (100 mM Tris-HCl pH 7.4, 50 mM EDTA pH 8.0, 0.5 % Triton X-100). The following primary antibodies were incubated overnight: OCT3/4 (1:100, sc-5279, Santa Cruz Biotechnology), ZSCAN4 (1:2000, AB4340, Millipore Sigma), γH2AX (1:1000, 05-636, Millipore), CTCF (1:1000, ab188408, Abcam), Flag (1:500, F1804, Sigma Aldrich). Corresponding Alexa Fluor 488 Chicken anti-Rabbit IgG (H+L) (Thermo Fisher Scientific, Cat# 31431), Alexa Fluor 488 Goat anti-Mouse IgG (H+L) (Thermo Fisher Scientific, Cat# A-11001), Alexa Fluor 647 Chicken anti-Rabbit IgG (H+L) (Thermo Fisher Scientific, Cat# A-21443) or Alexa Fluor 647 Chicken anti-Mouse IgG (H+L) (Thermo Fisher Scientific, Cat# A-21463) secondary antibodies were used to reveal primary antibody binding (1:1000). For generating the plots shown in Extended Data 2b, image analysis was performed using a custom Python script. In brief, DAPI-stained nuclei were segmented using the StarDist deep-learning image segmentation⁵² . Segmented nuclei ROIs were used to quantify total DAPI intensity and RFP mean intensity.

EDM cultures on coverslips were collected and fixed with 4% formalin for 30 min. All steps were performed at room temperature. EDMs were then permeabilised with PBS 0.2X Triton for 30 min and treated with NH₄Cl for 30 min to remove residual formalin. EDMs were incubated in PBS 1% BSA for 1 h, followed by overnight incubation at 4 °C with a rabbit anti-mouse cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, Danvers, MA, USA), or a mix of mouse anti-vertebrate γH2AX (Ser139) (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and rabbit anti-mouse 53BP1 (Novus Biologicals, Abingdon, UK) or rhodamine Phalloidin (Fischer Scientific). EDMs were then incubated for 2 h with a mix of Alexa Fluor 680 goat anti-mouse IgG (H+L) (Fischer Scientific) and Alexa Fluor 488 chicken anti-rabbit IgG (H+L) (Fischer Scientific) or with an Alexa Fluor Plus 680 Donkey anti-rabbit IgG (H+L) (Invitrogen, Waltham, MA USA, #A32802). EDMs were mounted in Prolong gold antifade mounting medium with 4′,6-diamino-2-phénylindole (DAPI) (Fischer Scientific) and examined under a Leica SP8 confocal microscope. Number of 53BP1 and γH2AX foci and cleaved caspase-3 fluorescence intensity were quantified with the ImageJ/Fiji software (version 1.54f).

MCF10A cells were fixed in 4% formaldehyde for 20 min, permeabilized in 0.15% Triton X‐100/PBS for 10 min and blocked in blocking solution (1% BSA, 0.05% Tween 20, PBS) for 30 min at room temperature. Primary antibodies diluted in blocking solution were added for 1 h at room temperature and detected by fluorochrome‐conjugated secondary antibodies. Nuclei were stained DAPI (Sigma–Aldrich). Primary antibodies used were anti‐ANGPTL4 (1:1000, rabbit monoclonal, Sigma–Aldrich), anti‐Fibronectin (1:1000, mouse monoclonal, Sigma‐Aldrich), anti‐FLAG M2 (1:250, mouse monoclonal, Sigma–Aldrich) and anti‐Rab8 (1:100, mouse monoclonal, Biolegend). Secondary antibodies used were Alexa Fluor 488 chicken anti‐mouse IgG (H+L) (1:400, Thermo Fisher Scientific), Alexa Fluor 488 chicken anti‐rabbit IgG (H+L) (1:400, Thermo Fisher Scientific), and Alexa Fluor 568 donkey anti‐mouse IgG (H+L) (1:400, Thermo Fisher Scientific).