Anti vegfr 3

Proteins were extracted from LMVECs after conditioning with 10% dcSSc serum (n = 8) or 10% healthy serum (n = 8) for 48 h, following previously published protocols [54 (link),55 (link)]. Thirty micrograms of total proteins were electrophoresed on NuPAGE 4 to 12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) and blotted onto polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked with a solution included in the WesternBreeze Chromogenic Western Blot Immunodetection Kit (catalog no. WB7105; Invitrogen) for 30 min at room temperature on a rotary shaker, and were incubated for 1 h at room temperature with the following rabbit polyclonal anti-human antibodies: anti-VEGFR-3 (1:1000 dilution; catalog no. ab27278; Abcam, Cambridge, UK), anti-NRP-2 (1:1000 dilution; catalog no. ab185710; Abcam), and anti-α-tubulin (1:1000 dilution; catalog no. #2144; Cell Signaling Technology, Danvers, MA, USA), assuming α-tubulin as control invariant protein. Immunodetection was performed using the WesternBreeze Chromogenic Western Blot Immunodetection Kit protocol (Invitrogen). Band intensities were quantified with the free-share ImageJ software, 64-bit Java 1.8.0_112 Windows version (NIH, Bethesda, MD, USA; online at http://rsbweb.nih.gov/ij), and the values were normalized to α-tubulin.

Immunofluorescence and phase-contrast images were used for analyzing the lymphatic vessels. For immunofluorescence staining, a macrofluidic device after experiments was fixed with 4% paraformaldehyde and incubated for 20 min at room temperature. Fixed cells were permeabilized using 0.1% Triton X-100 for 10 min. To prevent nonspecific binding, the samples were blocked with 1% BSA and incubated for 1 h at room temperature. Primary antibodies with 1% BSA, anti–VE-cadherin, anti-laminin, and anti-VEGFR3 (all from Abcam, United Kingdom) were filled into the channels and incubated for 2 h. After washing with 1x PBS three times, a mixture of Alexa Fluor conjugated goat anti-rabbit secondary antibody, rhodamine-phalloidin, and 4′,6-diamidino-2-phenylindole (all from Thermo Fisher Scientific) was filled in the device and incubated for 2 h. Images were obtained using a fluorescence microscope and a confocal microscope (LSM700; Carl Zeiss). A morphological analysis was conducted using ImageJ.