Rabbit anti mouse cleaved caspase 3 asp175 antibody

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).

Embedded thymi were sectioned (10 μm) using a cryostat. The slides were subsequently fixed with 4% paraformaldehyde for 20 min. Sections stained for cleaved caspase-3 were permeablized in a 0.1% citrate, 0.1% Triton X solution for 10 min. The slides were then washed 3 times in PBS, blocked in 5% donkey serum with 0.1% Triton X in PBS for 60 min, and then incubated with a 1:500 dilution of rabbit anti-mouse cleaved caspase-3 (Asp175) antibody from Cell Signaling (#9661) or with a 1:50 dilution of PE-conjugated anti-TIM4 (RMT4-54, eBioscience) diluted in PBS with 0.2% Triton X and 1% BSA overnight at 4 °C. The following day, Caspase-3 stained slides were then washed 3 times in PBS and incubated for 1 hour at room temperature with a donkey anti-rabbit Alexa488 fluorescently conjugated secondary antibody (Invitrogen). The slides were then stained with DAPI, washed 3 more times in PBS, and mounted with ProlongGold (Invitrogen). Images were acquired using an Olympus FV10i confocal microscope and analyzed using Imaris software (Bitplane).