Live imaging was carried out based on the protocol used in Ouspenskaia et al38 . E12.5 mouse embryos were placed on their sides in a 35-mm Lumox-bottom dish (Sarstedt). Each embryo was immobilized with a custom built holder and further stabilized in an agarose solution comprised of 2% low-melting SeaPlaque Agarose (Cambrex) in a solution of epidermal culture medium. After equilibrating at 37°C and 5% CO2 for ~2 hr, imaging was then performed on a custom-modified inverted spinning disk confocal system (Andor) at 12 min intervals for up to 24 hr (488 and 561 nm laser beams, 20× air objective (N.A.=0.75). Time-lapse images were acquired with a Zyla cCMOS camera (Andor). 3–4 regions were filmed from each embryo. During imaging, the embryo was maintained at 37°C and 5% CO2. Acquisition was controlled through iQ software (Andor).
All wholemount immunofluorescence images were acquired using a Zen-software driven Zeiss LSM 780 inverted laser scanning confocal microscope and either a 20x air objective (NA=0.8), a 40x water immersion objective (NA=1.2), or a 63x oil immersion objective (NA=1.4).
Images of epidermal cryosections were acquired using a Zeiss Axio Observer.Z1 epifluroescent/brightfield microscope with a Hamamatsu ORCA-ER camera, an ApoTome.2 slider (to reduce light scatter in z), and controlled via Zen software. All images were assembled and processed using ImageJ.
All wholemount immunofluorescence images were acquired using a Zen-software driven Zeiss LSM 780 inverted laser scanning confocal microscope and either a 20x air objective (NA=0.8), a 40x water immersion objective (NA=1.2), or a 63x oil immersion objective (NA=1.4).
Images of epidermal cryosections were acquired using a Zeiss Axio Observer.Z1 epifluroescent/brightfield microscope with a Hamamatsu ORCA-ER camera, an ApoTome.2 slider (to reduce light scatter in z), and controlled via Zen software. All images were assembled and processed using ImageJ.