Alexa488 azide

The senescence-associated beta-galactosidase staining (SAβG) was performed by using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA). After culture and fixation, the otic vesicles were washed in PBS and incubated in the X-Gal solution at pH 6 at 37°C protected from light. For SAβG in whole embryos at stages HH18 and HH19, the embryos were fixed for 15 min and incubated in the X-Gal solution. To perform the SAβG in chicken embryo tissue sections, the embryos were obtained, fixed for 15 min, frozen, and sectioned with a cryostat by the Histology Core of the “Centro Nacional de Biotecnología.” Sections were post-fixed for 2 min and incubated with the X-Gal solution.
For SAβG staining of otic vesicles, explanted otic vesicles were fixed with the fixative solution provided in the SAβG kit for 8 min and incubated with the X-Gal solution at pH 6 at 37°C. Then, otic vesicles were fixed with 4% PFA, permeabilized with PBS-T, washed with 2% PBS-BSA and incubated with the Click-iT Reaction Cocktail (Invitrogen) with Alexa-488 azide (5 μM, Invitrogen) at RT in darkness for 30 min. Finally, they were washed again with 2% PBS-BSA and mounted in Vectashield with DAPI (Vector, Peterborough, UK).

Monolayers of primary NSCs were exposed to 80 mM ethanol, and 12 h later, they were changed into fresh media containing 10 µM 5-ethynyl-2′-deoxyuridine (EdU; Invitrogen). The cells were fixed 1 h thereafter, permeabilized as above, and the EdU+ cells enumerated using Click chemistry with Alexa-488-azide (#A-11094; Invitrogen) diluted in 100 mM CuSO₄/100 mM ascorbic acid in Tris-buffered saline. Nuclei were visualized using DAPI. The omission of Alexa-azide served as a negative control. Images were captured under uniform exposure, capturing three images per sample.

Cells were seeded, labelled with thymidine analogues (CldU and EdU) and treated in the μ-slide 8 well chamber (ibidi, Gräfelfing, Germany, 80826). After treatments and labelling, cells were permeabilized for 10 minutes at 4 ºC with 0.5% Triton X-100 and fixed with 4% Paraformaldehyde containing PBS (131 mM NaCl, 1.54 mM KH2PO4, 5.06 mM Na2HPO4) for 10 minutes at RT. To denature DNA, samples were treated with 2N HCl dissolved in PBS containing 0.1% (v/v) Triton X-100 for 30 min at RT. For EdU staining, click reaction was performed during 30 minutes at RT with 1 μM Alexa488-azide (Invitrogen, A10266). Then, cells were incubated for 1 hour at RT with primary antibodies against CldU (Abcam; ab6326) and 53BP1 (Abcam, ab36823; 1/500) or γH2AX (Merck Millipore, Burlington, MA, USA 05636;1/3000) diluted in filtered DMEM: HAM’s F12 (1:1) supplemented as indicated in “Cell lines and culture” and 5% BSA. After a wash with PBS-T (PBS + 0.01% Tween20) cells were incubated overnight at 4°C with secondary antibodies. Finally, mounting media with DAPI (ibidi,50011) was added. Images were obtained with Zeiss LSM880 confocal microscopies (Confocal Microscopy Unit Core Facility, University of Barcelona) with a PLAN APO 63× oil immersion objective (numerical aperture 1.4) and analyzed using Fiji software. All data obtained were managed and analyzed with R studio software [51 , 52 ].