Fusion camera

Embryos and larvae were anesthetized in buffered 0.2 mg/mL Tricaine-S (Western Chemical, Inc; NC0872873) and mounted in 0.8% low-melting point agarose (IBI Scientific; #IB70056) on a 35-mm glass-bottom petri dish (MatTek; #P35G-1.5–20-C). Confocal images were obtained using a 4x or 40x objective with a W1 Spinning Disk confocal microscope, a Fusion camera, and the Nikon Eclipse Ti2-E base. Images were then deconvolved using Nikon’s NIS-Elements software. Fiji image processing software was used for counting number of cells per vessel length and for generating final images. Embryos were consistently imaged in the afternoon hours, thus: ‘1 dpf’ represents embryos between 28–34 hours post-fertilization (hpf); ‘2 dpf’ represents 52–58 hpf; ‘3 dpf’ represents 76–82 hpf; ‘4 dpf’ represents 100–106 hpf; ‘5 dpf’ represents 124–130 hpf. At least 6 embryos were imaged and analyzed per developmental timepoint.

HBVP cells were harvested and seeded at a concentration of 1.3 × 10⁵ cells in 0.4 optical plastic flow microslides (80176, Ibidi) precoated with 1 mg/mL gelatin and incubated for 24 hours under standard culture conditions. After the initial incubation, 100 μg/mL of collagen I (354249, Corning) diluted in DMEM cell culture media was added to the slides to create a thin layer on top of the HBVP cells. After 2 hours, the media was removed and 2.5 × 10⁵ HUVECs were seeded on top of the collagen I layer and incubated for additional 24 hours. After cell co-cultures were established, the slides were exposed to laminar (15 dyn/cm²) or pulsatile (12–15 dyn/cm²) flow for 24 hours, implementing a peristaltic pump adapted to produce different types of flow (Abello, Raghavan, Yien, & Stratman, 2022 (link)). After 24 hours, cultures were rinsed with 1x PBS and fixed for 30 minutes in 4% paraformaldehyde at room temperature. Cell cultures were immunostained with α-Smooth muscle actin D4K9N XP^® rabbit monoclonal antibody (19245, Cell Signaling), followed by Alexa Fluor 633 goat anti-rabbit IgG (A21071, Invitrogen). Confocal images were obtained using a 40x objective with a W1 Spinning Disk confocal microscope, a Fusion camera, and the Nikon Eclipse Ti2-E base. Fiji image processing software was used for image analysis and fluorescence intensity quantification.