Nunc glass bottom dish

Sorted cells were collected in 200 µl of 1× PBS and seeded in a Nunc glass-bottom dish (Thermo Fisher Scientific, #150680) previously treated with 200 µl of 20 µg ml⁻¹ fibronectin (Sigma-Aldrich, #F1141-2MG) overnight at 4°C. Cells were incubated for 3 h at 23°C, then 1× PBS was substituted with 200 µl growth medium and grown overnight at 23°C.
Cells were fixed for 5 min at room temperature with 4% formaldehyde in 1× PBS and washed once with 200 µl 1× PBS. Cells were then blocked for 30 min at room temperature in blocking solution [1% bovine serum albumin (Sigma-Aldrich, #A3294-10G), 0.1% Triton-X100 (Sigma-Aldrich, X100) in 1× PBS] and incubated for 1.5 h at room temperature with 1:100 anti-green fluorescent protein (GFP) primary antibody (Abcam, ab5450, Lot GR277059-1) in blocking solution [Venus is an improved version of GFP (Nagai et al., 2002 (link))]. Cells were washed twice for 10 min in blocking solution and incubated for 1.5 h in the dark at room temperature with 1:1000 Alexa Fluor 568 goat anti-rat IgG (Life Technologies, A11077, Lot 1512105) in the blocking solution. After three washes of 10 min in 1× PBS, the preparation was overlaid with fluorescence mounting media (DAKO/Agilent Technologies, #S3023), covered with a coverslip and sealed with nail polish.

The bright-field photographs of WISH, biochemical staining and blood were taken using a stereomicroscope (SMZ1500, Nikon). High-resolution images and movies of blood flow were taken under an upright microscope (Eclipse 80i, Nikon). Fluorescent images were taken using a confocal microscope (A1R+SIM, Nikon). Samples for microscopic observation and photography were prepared as previously described (Renaud et al., 2011 (link)). For live imaging, the embryos were anesthetized with 100 μg/ml tricaine, then embedded using 1.2% low melting agarose in a Nunc™ glass bottom dish (15068, Thermo Fisher Scientific).

THP‐1 cells were seeded on a Nunc glass‐bottom dish (Thermo Fisher Scientific) and incubated in RPMI 1640 medium at 37°C in a humidified atmosphere with 5% CO₂ overnight. The cells were washed with PBS three times after corresponding treatment, fixed with 4% paraformaldehyde for 15 min, and permeabilised with 0.25% Triton X‐100 for 10 min. After blocking with 5% skim milk in TBST for 30 min, the cells were incubated with anti‐LC3B (Cell Signal Technology, Danvers, MA, USA) for 2 h at room temperature, followed by incubation with Alexa Fluor^® 594‐conjugated anti‐Rabbit IgG (Thermo Fisher Scientific) for 1 h at room temperature. The nuclei were stained with DAPI for 5 min. After mounting, fluorescence images were acquired using a confocal laser‐scanning microscope (Zeiss, Jena, Germany). The experiments were repeated three times. A total of 100 cells were selected to calculate the average value at each step, and the average obtained from three independent experiments was plotted.