Ulex europaeus agglutinin 1

Mouse colon tissues (containing faecal pellet) were sampled and fixed with Cornoy solution (60% methanol, 30% chloroform, 10% glacial acetic acid) at 4 °C overnight. A tissue processor (Leica Microsystems) was used for paraffin embedding. Paraffin blocks were processed into thin sections (5.0 μm) using a microtome, followed by paraffin removal and immunostaining. The antibodies and stains used for immunofluorescence were as follows: rabbit anti-PRSS2 (LSBio, LS-C296077, 1:100), Alexa 488-labelled goat anti-rabbit IgG (Thermo Fisher Scientific, A11008, 1:1,000), 4′-6-diamidino-2-phenylindole (DAPI, DOJINDO), rhodamine-labelled UEA1 (Ulex Europaeus Agglutinin 1, Vector Laboratories). The Leica AF600 and confocal Leica TCS SP5 systems were used for immunofluorescence imaging.

Mature microvascular networks were rinsed with warm PBS followed by the addition of approximately 100 μl of 4% paraformaldehyde (Electron Microscopy Sciences, # 15700) to the media channels and left at room temperature. After 15 min of fixation, devices were rinsed twice with PBS, and blocking solution (4% bovine serum albumin, 0.5% goat serum) (Sigma-Aldrich) was added. Devices were incubated for 1 day at 4°C, washed with PBS, and stained with primary antibodies: ICAM-1 (Biolegend, 4453320),VCAM-1 (Abcam, ab134047), CD31 (Abcam, ab28364), conjugated Alexa Fluor 647 anti-human CD326 (EPCAM) (BioLegend, 324212), Acti-stain 555 phalloidin, F-actin (Cytoskeleton, PHDH1-A) and incubated at 4°C for another day. Devices were again washed with PBS and secondary antibodies (Thermo Fisher Scientific, A-11070, A-11011, A-21052) DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride, Invitrogen) or DyLight 649 labeled Ulex Europaeus Agglutinin I (Vector Laboratories) were added, followed by incubation at 4°C protected from light. Finally, samples were washed again with PBS and 3D images were acquired with a confocal microscope (Olympus FV1000) at 20×. Z-stacks were collapsed with maximum intensity projections for viewing (800 × 800 pixels) using FIJI (22 (link)).

Commercially available unconjugated and fluorochrome-conjugated Abs are listed in Supplementary Table 1. Chimeric, non-depleting, anti-α₄ integrin mAb (CRL19.11) was provided by R. Palframan. Anti-cytokeratin 8 mAb secreting hybridoma (TROMA-1) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa and PTX was from Calbiochem. Alexa Fluor® 488, Alexa Fluor® 647-labeled ovalbumin was purchased from Invitrogen and unlabeled EndoGrade ovalbumin was from Profos. High molecular weight fluorescein isothiocyanate and TRITC-dextran (MW = 2 × 10⁶ Da) were from Invitrogen. Fluorescently labeled Ulex Europaeus Agglutinin I was purchased from Vector Laboratories.

Co-cultured tissues in the device were fixed by aspiration of both reservoirs followed by 20 µL of 4% (w/v) paraformaldehyde (Biosesang, Korea) in PBS (Gibco, USA) in one reservoir per unit well with a 15 min incubation time bench top. Post fixation, both reservoirs were aspirated and optionally stored in 50 µL of dPBS per reservoir until ready for staining, or stained directly. Endothelial cell (EC) specific vessel staining was done with 488 fluorescein-labelled Ulex Europaeus Agglutinin I (Vector, UK), which was prepared at a 1:1000 ratio of dye in dPBS. Per unit well, 30 µL of dye solution was added to one reservoir and 10 µL to the other in order to facilitate the flow of dye from one reservoir, through the tissues, to the other. Samples were incubated at 4 °C overnight, then stored in 100 µL of dPBS per reservoir per unit well for imaging. Imaging was performed utilizing spinning disk confocal microscopy (Yokogawa CQ-1, Japan), and epifluorescence microscopy (Nikon TI-2, Japan) to produce to produce 3D and z-stackable images of the angiogenic and vasculogenic networks for figure generation and quantitative analysis of vasculature.
Given the standardized 384 well microtiter plate form factor of the platform, we utilized automated acquisition scripts to image entire 28 device chips utilizing imaging macros within both the Nikon and Yokogawa system.