Atto 633

hMSC were seeded as described in cell seeding. Two days after seeding, the cells were fixed with 4% paraformaldehyde/PBS for 5 min, washed once with PBS and stored in PBS at 2–8 °C before staining. The samples were then washed 3 times with PBS and incubated in 3% FBS/0.1% Triton X in PBS for 30 min at RT. The samples were then incubated with 1% Tween 20 in PBS for 20 min at RT. Monoclonal antibody against talin (dilution 1 : 200, clone 8d4, ascites fluid, T3287, Sigma-Aldrich) or vinculin (dilution 1 : 100, monoclonal hVIN-1, product V9131, Sigma-Aldrich) was applied overnight at 2–8 °C. After washing 3 times with PBS/0.05% Tween (3, 5, 10 min) and once in PBS, anti-mouse secondary antibody conjugated with goat anti-mouse IgG/IgA/IgM (H + L) Alexa Fluor 488 (dilution 1 : 300, A10667, Invitrogen Life Technologies) was added for 45 min at RT. Subsequently, after same washing procedure, the samples were incubated with phalloidin conjugated with ATTO-633 (dilution 1 : 50 in 1 mL PBS, Sigma Aldrich) for 1 hour at RT. Cell nuclei were counterstained with Hoechst 34580 (1 : 5000, H21486, Life Technologies) for 30 min at RT and washing twice in PBS. The samples were scanned using confocal microscope ZEISS LSM 5 DUO at wavelengths described above, and λ_ex = 633 nm, λ_em >650 nm for ATTO-633 staining.

To analyze overall nanoparticle distribution, cell membranes were mosaically labeled by co-injecting Tol2-Ubi-mKate-CAAX plasmid DNA (courtesy of Sara Caviglia). Liver progenitors were visualized by the immunostaining for EfnB1^{24 (link)}.
To determine the precise location of nanoparticles used for measuring viscoelasticity, the embryos were fixed in 4% paraformaldehyde (PFA) immediately after the optical trapping experiments, deyolked, and stained with phalloidin conjugated to Atto 633 (Sigma-Aldrich, USA) to visualize the actin cell cortex and tissue morphology. The foregut region was imaged using a Leica SP8 confocal microscope. Images obtained from confocal scanning simultaneously with the trapping experiments were used to identify the particles of interest in the fixed sample by manually correlating foregut shape and nanoparticle distribution in 3D using Bitplane IMARIS software. The nanoparticles were assigned to a specific cell population based on tissue morphology and transgenic sox17:GFP expression. Only single beads, which could be clearly assigned to a specific tissue, were included in the analysis.