Rat anti mouse ve cadherin

Murine or human LEC monolayers were stained for surface VCAM-1, VE-Cadherin or Lyve-1 with rat anti-mouse VCAM-1 (Clone 429, eBioscience) or mouse anti-human VCAM-1 (Clone STA, Biolegend), rat anti-mouse VE-Cadherin (Clone 11D4.1, BD Biosciences) or mouse anti-human VE-Cadherin (Clone BV9, Biolegend), goat-anti-mouse or anti-human CCL21 polyclonal antibody (R&D System), or rabbit anti-mouse Lyve-1 (70R-LR003, Fitzgerald, Acton, MA), then were fixed for 20 min at 4 °C with 4% (w/v) paraformaldehyde (Affymetrix, Santa Clara, CA), Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), and treated with 5% donkey serum for 30 min then incubated with listed primary antibodies for overnight at 4 °C. Samples were incubated with secondary antibodies conjugated with Alexa Fluor 448, 546 or 647 (1:400, Jackson ImmunoResearch, West Grove, PA) for 1 hour at 4 °C and mounted in Prolong Gold with DAPI (Thermo Fisher, P36931). Transwell membranes were transferred onto glass slides and visualized by fluorescent microscopy (Zeiss LSM 510 Meta and LSM5 Duo) with a 60× or 20× objective. Images were analyzed with Volocity version 6.3 software. Fluorescence intensity was measured as Mean Fluorescence Intensity (MFI) over the entire field.