Plan apochromat 40x 1.3 oil

Cells were seeded on top of a collagen I matrix and immunostained as described^{12 (link)}. Cells were fixed with 4% formaldehyde, permeabilised with 0.3% Triton X-100, blocked with 5% bovine serum albumin (BSA), and stained with primary antibody pSer19-MLC2 (1:200, #3671, Cell Signalling), which was detected with secondary Alexa Fluor 488 or 647 antibodies (Life Technologies). F-actin was stained using Alexa Fluor 546-phalloidin (Life Technologies) and nuclei with Hoechst 33258 (Life Technologies). Antibodies were diluted in 5% BSA-PBS. Imaging was carried out on Zeiss LSM 510 Meta confocal microscope with Plan-Apochromat 40x/1.2 NA (water) objective lenses and Zeiss LSM 710 confocal microscope with Plan-Apochromat 40x/1.3 Oil or a Plan-Apochromat 63x/1.4 NA (oil) objective lenses (Carl Zeiss) and Zen software. Images were analysed using ImageJ. p-MLC2 fluorescence signal was quantified calculating the pixel intensity in single cells relative to the cell area.

Microscope images for fixed and stained Drosophila follicles were taken using LAS SPE Core software on a Leica TCS SPE mounted on a Leica DM2500 using ACS APO 20x/0.6 IMM Corr -/D objective (Leica Microsystems), LAS-X software (SCR_013673) on a Leica DMi8 Stellaris using a HCPLAPO CS2 20x/0.75 Dry and HCPL APO CS2 63x/1.4 Oil, Zen software (SCR_013672) on Zeiss 700 LSM mounted on an Axio Observer.Z1 using a Plan-Apochromat 20x/0.8 M27 or EC-Plan_Neo_Fluar 40x/1.3 Oil, Zeiss 880 mounted on Zeiss Axio Observer.Z1 using Plan-Apochromat 20x/0.8, Plan-Apochromat 40x/1.3 oil (Carl Zeiss Microscopy), or NIS-Elements Software (SCR_014329) on Nikon ECLIPSE Ti2-E inverted microscope using Plan Apo λD 20x/0.8 Dry. S9 follicles were identified by their size (∼150 μm–250 μm) and morphology, including, the location of the outer follicle cells and the border cell cluster. The beginning of S10A was defined as when the anterior most outer follicle cells reached the nurse cell-oocyte boundary and flattened. Maximum projections, merge, rotation, and cropping were performed using ImageJ software (FIJI, RRID: SCR 002285 (Abramoff et al., 2004 )). All images shown were brightened by 30% in Photoshop (Adobe, RRID: SCR 014199), except where noted, to improve visualization and figures were made using Illustrator (Adobe, RRID: SCR 010279).