Fluorescent carboxylate microspheres

3D-liver models were fixed with 4% formaldehyde for 30 min. Blocking and incubation with the first antibody was (overnight in PBS containing 1% BSA) was followed by 2 h of incubation with secondary antibodies (Cell Signalling) in the same buffer. Immunostaining was performed with antibodies specific for human integrin-β1 (Millipore, 3199Z), ICAM-1 (R&D systems, 11C81) and E-cadherin (Cell Signaling, 24E10). Images were acquired with a multiphoton LSM710_NLO upright microscope, using the multi-photon laser and SHG to visualize the COL-I matrix, or the confocal laser the reflection mode to visualize the COL-I matrix and cell topography.
To determine the intensity of E-cadherin labelling, 1 µm diameter fluorescent carboxylate microspheres (Polysciences, 15702) were used as reference. Fluorescence intensities of E-cadherin signals and the beads was measured using Zen software from Zeiss. The intensity of the E-cadherin label was normalized by the intensity of the beads (added prior to microscopy) present in the same field and focal plane (set as 100%).

Permeability of LSEC monolayers or the COL-I matrix was determined through the ability of 1 µm diameter fluorescent carboxylate microspheres (Polysciences, 15702) to pass through the cell layer or the matrix border. After 3 h of incubation, the beads added on top of the samples, the COL-I matrix and the cells were visualized by two-photon microscopy. For each microscopic field (0.28 mm²) the x-, y- and z-position of each fluorescent bead and the z-position of the cell layers or the upper matrix border were determined using ICY software (http://icy.bioimageanalysis.org). A LabMat script was created to represents the z-stack position of individual beads, with the matrix border or the LSEC layer position set to 0 µm.