Microscopy slides

For each examined species, all samples were collected from the same egg clutch. Sampling covered the entire period of somitogenesis, which coincides with NC development, with samples taken at 3-hour intervals. At each time point, at least four embryos were dissected and placed individually into either 250 μl of pre-chilled Trizol (Ambion) and stored at −80°C until RNA extraction (at least overnight) or into 1 ml of 4% PFA in 1X PBS for overnight fixation at 4°C. Embryos preserved in 4% PFA were later rinsed twice in 1X PBS and stained with 10 nM DAPI in 70% glycerol in 1X PBS overnight at 4°C, protected from light. Following a wash in 1X PBS (10 min/wash, once), the embryos were mounted on microscopy slides (ThermoFisher) with Fluoromount G (Southern Biotech) and imaged with an Olympus FV3000 confocal microscope to confirm the developmental age (somite stage) of each sampled cohort.

After exposure to 1, 10, 20 and 40 µM SM or 200 µM H₂O₂, senescence development was checked every third to fourth day using the senescence detection kit I (PromoCell, Heidelberg, Germany) according to manufacturer’s protocol. To exclude false positive staining, cells were plated at the same density 1 day before staining onto cover slips in 4-well plates. Briefly, 10,000 cells were grown overnight on coverslips in 4-well plates, washed once with PBS and fixed for 10–15 min at room temperature with the fixative solution. Meanwhile, the senescence staining solution was prepared by mixing 470 µL of staining solution, 5 µL of staining supplement and 25 µL of 20 mg/mL X-gal in DMSO per well. Fixed cells were washed twice with PBS and senescence staining solution was added and developed in the incubator overnight. Additionally, cells were counterstained with nuclear fast red (Vector Laboratories, Inc., Burlingame, USA) for 10 min at room temperature and mounted with VectaMount™ AQ mounting medium (Vector Laboratories, Inc., Burlingame, USA) onto microscopy slides (Thermo Fisher Scientific, Waltham, USA). Senescence development was monitored over a time period of 4 weeks. The percentage of senescent versus total cells was counted in three randomly selected images (mean of 20 cells per image) and was performed in three independent experiments.

For the analysis of resting B cells, wells of the microscopy slides (10028210, Thermo Fisher Scientific) were coated with CellTak (354240, Corning) in PBS (3.5 μg/cm² of surface area, according to manufacturer's recommendations) for 20 min (RT), washed once with water and allowed to dry. For the analysis of activated B cells, wells were coated with 5 μg/ml of anti-IgM in PBS for 1 h (RT) and washed in PBS. 10⁵ B cells in 20 μl of complete RPMI were placed on coated wells for 10 min (+37°C, 5% CO₂) and fixed by adding 20 μl PFA in PBS (4% PFA final, pH 7.0–7.5) for 15 min. Samples were further fixed in 4% PFA/2.5% gluteraldehyde in PBS for 30 min, washed in PBS and post-fixed in 1% OsO₄ containing 1.5% potassium ferrocyanide, and dehydrated with a series of increasing ethanol concentrations (30, 50, 70, 80, 90, 96, and twice 100%). Specimens were immersed in hexamethyldisilazane and left to dry by solvent evaporation. The cells were coated with carbon using Emscope TB 500 Temcarb carbon evaporator and imaged with Leo 1530 Gemini scanning electron microscope.