Ec plan neofluar 40x na1.3 wd0 17mm

Immunolabeled embryos were mounted on 35 mm glass-bottom dishes (MatTek), within micro drops of a 5 µg/ml solution of Hoechst 33342 in PBS and imaged using a Zeiss LSM880 laser-scanning confocal microscope, equipped with an oil-immersion Zeiss EC Plan-Neofluar 40x/NA1.3/WD0.17mm. Z-stacks were acquired through whole embryos with 1 µm step between optical slices. Laser power was measured for each laser line prior to each imaging session and parameters adjusted so as to keep laser power consistent for each primary-secondary antibody combination across experiments over time – except for the 405 channel, which was used to excite the nuclear label (Hoechst 33342), and was solely used for image segmentation.

Embryos were cultured on 35 mm Petri dishes (Falcon) within microdrops (10–15 µl) of Potassium Simplex Optimized Media with amino acids (KSOM-AA, Millipore) under mineral oil (Sigma), at 37°C, in a humidified 5% CO₂ atmosphere. Prior to culture, embryos were rinsed 3x in drops of KSOM-AA. For live imaging, embryos were cultured on 35 mm glass-bottom dishes (MatTek) within 5–10 µl drops of KSOM-AA under mineral oil.
Images of live embryos were acquired using Zeiss LSM880 laser-scanning confocal microscopes. In cell ablation experiments, images were acquired using a Zeiss C-Apochromat 40x/NA1.1/WD0.21mm objective. For all other experiments, images were acquired using a Zeiss EC Plan-Neofluar 40x/NA1.3/WD0.17mm. GFP was excited using a 488 nm Argon laser at 20µW. mKate2 was excited using a 543 nm HeNe laser or a 561 nm DPSS 561–10 laser at 90µW. Laser power was measured through a Zeiss Plan-Neofluar 10x/NA0.3 objective prior to each imaging session with a light meter (Coherent) and the laser output adjusted to match laser power across experiments. 80 µm stacks were acquired through embryos, at 2 µm intervals, every 15 min. Although these stacks do not capture an entire blastocyst, they encompass the ICM while limiting laser exposure to about 30–40 s per time point and embryo. Time lapse movies were 16–20 hr long.