Anti lyve 1

Corneas were removed and immediately fixed with acetone, followed by blocking in 2% BSA. Corneas were stained with Alexa Fluor-647 anti-vimentin (Abcam, Cambridge, UK), anti-F4/80 (Invitrogen, Eugene, USA), anti-NFAT5 (Thermo Fisher, USA) or anti-LYVE1 (Abcam, UK). Secondary antibodies for staining included Alexa Fluor 555 goat anti-rat and Alexa Fluor 488 goat anti-rabbit (Invitrogen, USA).

Doxycycline, hydroxypropyl-β-cyclodextrin, poloxamer 407, poloxamer 188, VEGF-C and lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO, USA). Antibodies included, anti-LYVE-1, anti-VEGF receptor 3 (VEGFR3) (abcam, Hong Kong, China), anti-Akt, anti-phosphorylated Akt, anti-nuclear factor-kappaB (NF-κB) p65, anti-phosphorylated NF-κBp65, anti-IκB-α, anti-eNOS, anti-phosphorylated eNOS, anti-β-actin, horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, HRP-conjugated anti-rabbit secondary antibody, Alexa Fluor 488-coupled goat anti-rat secondary antibody and Alexa Fluor 555-coupled goat anti-rabbit secondary antibody (Cell Signaling Technology, Inc., Danvers, MA).

Popliteal lymph node metastasis assay was performed as discribed in the paper of Libing Song and their colleagues [32 (link)]. Briefly, BALB/c-nu mice (female, 5–6 weeks old, 18–20 g) were randomly divided into two groups (n = 9/group). The miR-548k and GFP stable expression KYSE30 cells (5 × 10⁵) were inoculated into the foot-pads of the mice. The mice were sacrificed after half and a month, and the primary tumors and popliteal lymph nodes were collected and paraffin embedded. Serial 4.0 μm sections were taken and analyzed by IHC with anti-LYVE-1 (Abcam) and anti-GFP (Santa Cruz) antibodies.

The following antibodies were used in immunoblotting: anti-E-cadherin (Cell Signaling, 3195, 1:1000), anti-Zeb1 (Santa Cruz, sc-25388, 1:1000), anti-Beta-Actin (Cell Signaling, 4970S, 1:1000), and anti-Gamma-Tubulin (Sigma, T6557, 1:5000). The following antibodies were used for immunohistochemistry: anti-SV40 T Ag (Santa Cruz, sc-20800, 1:50), anti-Insulin (DAKO, A056401, 1:1000), anti-CD31 (R&D Systems, AF3628, 1:15), anti-Lyve1 (Abcam, ab14917, 1:100), anti-E-cadherin (Abcam, ab76055, 1:200), anti-Glucagon (Millipore, AB932, 1:200), anti-SMA (Abcam, ab5694, 1:100), anti-CK19 (Abcam, ab52625, 1:400).

The heart and aorta were removed and fixed in 4% PFA for 2 hours.The heart was transferred into PBS containing 30% sucrose (wt/vol) overnight at 4 °C before being immersed in OCT compound and stored at −80 °C. Eight-micrometer-thick cryosections of the aortic sinus were prepared. Cross-sections of the aortic sinus were stained with anti-LYVE-1 (ABCAM) and anti-CD68 (Biolegend) antibodies, and then incubated with the appropriate secondary antibodies. As macrophages can also be positive for LYVE-1, adventitial lymphatic capillaries were identified as LYVE-1⁺ CD68⁻ cells forming vessel-like shapes. Whole-mount immunohistochemical analysis of the ear dermis to visualize lymphatic vessels was performed as described previously47 (link). Ear dermis were stained for lymphatic capillaries (anti-LYVE-1, ABCAM) at 4 °C, and then sections were incubated with Alexa Fluor 647 conjugated donkey anti-rabbit antibody and Cy3 donkey anti-rat (Jackson ImmunoResearch). All imaging was performed on a Fluoview FV10i (Olympus). All vessel counts were performed by one observer. The relative quantification of the number of lymphatic capillaries (LYVE-1⁺ vessels), their diameter and the total surface area they occupy was determined by computer-assisted morphometric analysis. Neutral lipid assessment in atherosclerotic lesions was performed by Oil-red-O (ORO) staining (Sigma).

Heart allografts and LNs were harvested at designated time points after transplantation. Heart allografts were fixed in formalin, embedded in a paraffin block, or preserved in optimal cutting temperature (OCT) compound (Tissue-Tek) and stored at –80°C. Samples were cut into 5-μm sections and stained with H&E. For immunofluorescent staining, sections were stained with conjugated or purified antibodies. Purified antibodies were detected using secondary antibodies. The antibodies included anti-CD11b (BioLegend, 101202), anti–collagen I (Abcam, ab34710), anti–Lyve-1 (Abcam, ab14917), anti-ERTR7 (Santa Cruz Biotechnology, sc73355), anti-fibronectin (Abcam, ab2413), anti-Foxp3 (BioLegend, 126401), and anti-CD3 (BioLegend, 100201). DAPI (VECTASHIELD, Vector Laboratories) was used to counterstain the cell nuclei. The stained tissue sections were visualized using an EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific). Quantification was performed on 4 to 5 sections from at least 3 separate mice using Celleste (Invitrogen) and ImageJ (NIH, v1.8.0_112) image analysis software.