Apotome 2 slider

Cells were incubated with 1 μg/ml DAPI (Sigma Aldrich) and 7.5 μM Draq5 (ThermoFisher Scientific) at RT for 10 and 25 min, respectively. Cells were fixed by adding 37% formaldehyde (final 3.7%) into supernatant HSC medium and incubated for 1 h at RT. Next, samples were washed 3x with PBS and incubated for 1 h at RT in blocking solution (BS: 1% BSA, 0.4% Triton-X 100 in PBS). Samples were incubated in anti-human CD34 primary antibody (Santa Cruz Biotechnology, 1:250 in BS) overnight at 4°C. Afterwards, samples were 3x times with PBS and incubated for 2 h at RT in AlexaFlour^®555-conjugated anti-mouse secondary antibody (Abcam; 1:500 in BS). Finally, samples were washed 3x with PBS before mounting with ProLong™ Gold Antifade Mountant (ThermoFisher Scientific). Z-stack images were taken with a Zeiss Axio Imager microscope equipped with an ApoTome.2 slider (Carl Zeiss) as well as with a Zeiss Axio Scan.Z1 microscope (Carl Zeiss) and analyzed using Zen 2.0 software (Carl Zeiss).

Images and image stacks of sections were acquired using an Axio Imager. Z1 equipped with an ApoTome.2 slider for optical sectioning (Zeiss, Germany). For image processing the ZEN2 blue software (Zeiss, Germany) was used and either single images or extended depth of focus projection of z-stacks were displayed. Whole mount larvae were either imaged as described (α-NeuN staining) or with a light sheet microscope (α-Cldn5 staining, morpholino experiments). For light sheet microscopy, a Lightsheet Z1 (Zeiss) enabled for dual side illumination and equipped with a 20x detection objective (W Plan-Apochromat, numerical aperture = 1.0) and a sCMOS pco. edge 4.2 camera was employed. Image processing consisting of dual side fusion, brightness/contrast adjustment and unsharp masking was performed by using the Zen 3.1 (blue edition) software (Zeiss, Germany). For three-dimensional reconstruction the 3Dxl rendering module (powered by arivis, Germany) was used.