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.