C apochromat 40x 1.2 w korr m27

Procedures for immunofluorescence staining including foci detection of single cells and FFPE tissue sections were described previously [14 (link), 17 (link)]. Antibodies are listed in the Suppl. Table S2. Tissues were visualized on an LSM 710 (Carl Zeiss GmbH) using an EC Plan-Neofluar 10x/0.3, or C-Apochromat 40x/1.2 W Korr M27 objective. Standard hematoxylin–eosin (HE) staining was visualized on Echo Rebel REB-01 hybrid microscope (20x/0.40 LWD Achromat Phase objective).

Samples were imaged on a Zeiss Airyscan LSM 880 confocal microscope with a C-Apochromat 40x/1.2 W Korr M27 water immersion objective or a Plan-Apochromat 63x/1.4 OIL DIC M27 objective. For super-resolution imaging, an Airyscan detector was used⁷⁹ . Volocity (version 6.3.1, PerkinElmer) and Zen (Zeiss) software were used to produce maximum intensity projections and 3D opacity renderings. Image analysis was performed on optical sections. For signal intensity profiles along the apical-basal axis and across tricellular junctions, the arrow tool in the Zen software was used. Anterior and posterior embryo widths measurements were made using the line tool in Volocity.
For F-actin signal intensity profiles across the apical surface of epiblast or ectoderm cells, Fiji’s freehand line tool with a width of ‘3’ was used^{80 (link)}. Because the size of the apical domain was different for each cell measured, distances were expressed as percentages, with 100% representing the total distance across the apical domain. To account for depth-dependent signal attenuation, F-actin signal intensity at the apical domain was normalised by mean F-actin intensity in the nucleus of the cell measured. In each experiment, for each genotype, three embryos were used for measurements and five cells were analysed per embryo. The LOWES method was used to fit a line to the data.