Axiocam mrc5 digital

For the LIVE/DEAD viability/cytotoxicity assay (Life Technologies), aHSCs and rHSCs were incubated with PBS containing 4 μM ethidium homodimer and 2 μM calcein for 15 min at 37 °C, washed thoroughly with PBS, and subsequently analyzed under a fluorescent microscope (Carl Zeiss). Images were taken with an AxioCam MRc5 digital camera (Carl Zeiss).

Embryos were anesthetized with 0.03% tricaine (Sigma-Aldrich, Saint Louis, MO, USA) and mounted in 4% methylcellulose. All images were captured by a Zeiss Axio Imager Z1 microscope with the AxioCam MRc5 digital camera (Zeiss, Oberkochen, Germany) and processed by Adobe Photoshop CC 2018.

Tg (flk1:eGFP) transgenic zebrafish were bred and maintained in normal condition (28°C; pH 7.2‐7.4; 14 h on and 10 h off light cycles). A total of 5‐10 nL suspension containing 10 ng ctrl‐Morpholino (MO) or IMD‐MO were injected into zebrafish embryo 48 h post fertilization through the perivitelline space in a single injection by using an electronically regulated air‐pressure microinjector (Harvard Apparatus, NY, PL1‐90). For each implantation, about 50‐60 fish were transferred to six‐well plate containing 2 mL of fresh fish water. In the IMD rescue group, zIMD peptide (synthesized mature zebrafish‐IMD, VGCVLGTCQVQNLSHRLYQLVGQSGREDSPINPRSPHSY) was added into the fish water to reach a final concentration of 2 µM. The fish water was changed daily.
The living zebrafish embryos were anesthetized using 0.003% tricaine and embedded in 3% methylcellulose. The digital micrographs were acquired using Zeiss Imager Z1 fluorescence microscope (Carl Zeiss Microimaging Inc, Germany) equipped with an AxioCam MRc5 digital CCD camera or a Zeiss LSM 510 Meta Confocal Microscope. Images were taken in the same focal plane in brightfield and transmitted light passing through GFP filters (488 nm); 0.5‐2 µm step z‐stacks (512 × 512 focal planes, 50‐200 µm in depth) were acquired by using 10× or 20× objective lens. Image capture and processing were performed by using ZEISS Axiovision rel.4.8 software.