αrabbit af488

The cell culture medium was aspirated and 75 μL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (J61899, Alfa Aesar, USA) was added to all the perfusion inlet and outlet wells [10 (link)]. The device was placed on a rocker to induce flow and incubated overnight at 4°C. After fixation, the PFA was aspirated from the wells and the microfluidic chips were washed three times with 0.3% Triton X-100 in PBS in all the perfusion inlets and outlets, followed by a permeabilization step of 1% Triton-X100 (VWR 28817295). Nuclei were stained using 1:2000 Hoechst 33258 (H3569, Life Technologies, USA). Tissue construct was incubated with primary antibodies for overnight; 10 μg/mL αβ3-tubulin, AF488 (Invitrogen, 53-4510-82), 0.36 μg/mL αCD31, Rabbit (Invitrogen, PA5-16301), 5 μg/mL αPECAM1 (Millipore Sigma, WH0005175M1), 10 μg/mL αVE-Cadherin, Rabbit (Sigma-Aldrich, V1514), and 5 μg/mL αZO- 1, Mouse, AF488 (Invitrogen, 339188). Secondary antibodies with blocking solution are further incubated; 2 μg/mL αMouse, AF568 (Invitrogen, A-11004), 4 μg/mL αRabbit, AF568 (Invitrogen, A-11036), and 2 μg/mL, αRabbit AF488 (Invitrogen, A-11008). Image data was acquired and processed using confocal microscope (ZEISS Multiphoton LSM 710) and ZEN software. Imaging depth was set at 16 bits, binning at 1, and imaging resolution at 2048 × 2048. Autofocus was set at a 120 μm offset from channel bottom.

Tissue construct was extracted from the OrganoPlate Graft. Tissues were fixed in 4% paraformaldehyde overnight at 4°C followed by washing in PBS 3 times for 5 min. Tissues were allowed to sink in 30% sucrose overnight and then embedded in OCT, flash frozen, and cryosectioning at 14 μm. Section were blocked and permeabilized in 0.2% Triton-X in PBS. Sections were then incubated with primary antibodies in 0.1% Tween-20, 10% normal donkey serum at the following dilutions: 0.3 μg/mL βIII-tubulin, Chicken (Millipore Sigma, AB9354), 10 μg/mL αTBR1, Mouse (Proteintech, 66564-1-Ig), 2 μg/mL αSATB2, Rabbit (Invitrogen, PA5-83092), 2 μg/mL αN-Cadherin, Rabbit (Invitrogen, PA5-85495), and 4 μg/mL αβ1-Integrin, Mouse, AF647 (Santa Cruz Biotech, sc-9970). Nuclei were stained using 1:2000 Hoechst 33258 (H3569, Life Technologies, USA). Secondary antibodies are further incubated; 3 μg/mL αMouse, AFP488 (Invitrogen, A32766), 3 μg/mL αRabbit, AF568 (Invitrogen, A-11036), 5 μg/mL αRabbit, AF488 (Invitrogen, A-11008), 5 μg/mL αChicken, AF647 (Invitrogen, A-21449), and 5 μg/mL αChicken, CF594 (MilliporeSigma, SAB4600094).