Lyve 1

The primary antibodies VEGF-D, LYVE-1, β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); VEGFR-3/phospho-VEGFR-3, JNK phospho-JNK, Bcl-2 and Bax antibodies were purchased from Cell Signaling Laboratories (Beverly, MA). The terminal deoxynucleotidyl transferase mediated nick-end labeling (TUNEL) assay kit was purchased from Promaga Company (Madison, WI). DMSO, Tween-20, Gelatin and CMC-Na were purchased from Sigma Chemical Co, (St. Louis, MO); Matrigel was purchased from BD Pharmingen (La Jolla, CA).

LGs were harvested and analyzed by immunohistochemical (IHC) staining and immunofluorescence staining. Histologic sections (5 to 7 μm) were collected on poly-L-lysine-coated slides and deparaffinized. The sections were then rehydrated with a xylene-grade alcohol scale and rinsed with phosphate-buffered saline. Sections were blocked with rabbit/goat/rat serum for 40 minutes at room temperature and exposed to primary antibodies: NOTCH1 (Goat polyclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc., Dallas, TX), DLL4 (Rabbit polyclonal anti-mouse, 1μg/ml; Abcam^®, Inc.), LYVE-1 (Rat monoclonal anti-mouse, 2μg/ml; Santa Cruz Biotechnology, Inc.). Antibodies were diluted from 1:100 to 1:200 and incubated overnight at 4°C. After washing in Tris-buffered saline supplemented with Tween 20 (TBST), each section was exposed to secondary antibodies for 1 hour. After washing out the secondary antibodies with TBST, the sections were exposed to 4',6-diamidino-2-phenylindole (DAPI) (PureBlu™, Bio-Rad, Inc., Hercules, CA). The IHC staining method for LG has been described previously.[11 (link)] Anti-CD45 antibody (Rabbit polyclonal anti-mouse, 0.5 μg/ml; Abcam^®, Inc.) was used for IHC staining of inflammatory cells. Light microscopy (Axio Imager 2, Carl Zeiss, Germany) was used for examination.

The sources and characteristics of the fucoidan used in this study have been previously reported [13 (link)]. Fucoidan was diluted in Roswell Park Memorial Institute 1640 (RPMI 1640) complete medium at a concentration of 800 μg/ml. RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, South America). Trypsin was obtained from Hyclone (Thermo Fisher Scientific, Carlsbad, CA, USA).
The following primary antibodies were used in this study: VEGFR3, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and phospho-Akt (p-Akt; Santa Cruz, CA, USA); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclin-dependent kinase 4 (CDK4; Sangon Biotech, Shanghai, China); PROX1 and cyclin D1 (Boster, Wuhan, China); NF-κB (Zhongshan Biotech, Beijing, China) and p-PI3K (ImmunoWay, Newark, DE, USA).