Vegf c

Tissue sections were incubated with 3% H₂O₂ and 10% methanol in 0.01 M phosphate buffered saline (PBS; Sigma-Aldrich) to destroy endogenous peroxidase activities. Next, sections were blocked with 10% normal goat serum in 0.01 M PBS for 1 h and then incubated overnight at 4℃ with antibodies to VEGF-C (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGFR-3 (1:500; Abnova, Taipei, Taiwan), glial fibrillary acidic protein (GFAP, 1:400; Chemicon International Inc., Temecula, CA, USA), or Ox42 (1:100; Serotec, Oxford, UK). On the next day, sections were incubated with anti-rabbit immunoglobulin G (IgG) (1:200), anti-mouse IgG (1:500), or anti-rat IgG (1:500) for 2 h at room temperature. Finally, the sections were visualized with 0.1% diaminobenzidine tetrahydrochloride and 0.005% H₂O₂ in 0.05 M Tris HCl (pH 7.4) and observed using a light microscope (BX51; Olympus, Tokyo, Japan).
For triple labeling, the sections were incubated with rabbit anti- VEGF-C or VEGFR-3 (1:500), mouse anti-GFAP (1:400), and rat anti-Ox42, followed by Cy3- (1:500; Jackson ImmunoResearch, West Grove, PA, USA), Cy5- (1:500; Jackson ImmunoResearch), and Alexa fluor 488-conjugated IgG (1:300; Invitrogen, Carlsbad, CA, USA), respectively. Finally, sections were mounted and observed using a confocal microscope (LSM 510 Meta; Carl Zeiss).

Cells were harvested and homogenized with lysis buffer (Beyotime Institute of Biotechnology) 48 h post-transfection. Protein concentrations were measured using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (between 30 and 50 µg) were separated by SDS-PAGE (10% gel) and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Subsequent to blocking with 5% skimmed milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies specific to MTDH (catalog no., 14065S), vascular endothelial growth factor C (VEGF-C; catalog no., 2445S), cyclin D1 (catalog no., 2978T), epithelial (E-) cadherin (catalog no., 3195S) and β-actin (catalog no., 3700S) (all dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA). The membranes were then incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse secondary antibody for β-actin; dilution, 1:5,000; catalog no., sc-2005 or mouse anti-rabbit secondary antibody for MTDH, VEGF-C, cyclin D1 and E-cadherin; catalog no., sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The resulting immunoblots were quantified using ImageJ2x software (National Institutes of Health, Bethesda, MD, USA).