Immunohistochemistry. Immunohistochemistry was performed on cultured human lymphatic endothelial cells (LECs) or fresh-frozen sentinel lymph nodes (SLNs) isolated from breast cancer patients. In brief, the cultured LECs were fixed with 10% formalin in phosphate-buffered saline solution (PBS) at room temperature. The cells were incubated for 4 h at room temperature with primary polyclonal antisera platelet–endothelial cell adhesion molecule (PECAM)-1 (dilution 1:100, BD Biosciences, Franklin Lakes, NJ, USA), lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 (dilution 1:50, RELIATech, Braunschweig, Germany), Prox-1 (dilution 1:50, AngioBio, Del Mar, CA, USA), podoplanin (dilution 1:50, AngioBio), vascular endothelium growth factor receptor 3 (VEGF R3) (dilution 1:50, Santa Cruz, Santa Cruz, CA, USA), E-selectin/CD62E (dilution 1:50, R & D Systems, Minneapolis, MN, USA), P-selectin/CD62P (dilution 1:50, R & D Systems), vascular cell adhesion molecule (VCAM)-1/CD106 (dilution 1:50, R & D Systems), and intercellular adhesion molecule (ICAM)-1/CD54 (dilution 1:50, R & D Systems). Before staining, permeabilization of the cultured cells with 0.1% Triton-X was performed. Then, the cells were stained using the following antibodies: Alexa Fluor 488 chicken antirabbit IgG or Alexa Fluor 488 donkey antimouse IgG (Invitrogen, Carlsbad, CA, USA). The nuclei of cultured cells were counterstained and mounted with ProLong Gold antifade reagent with 4′-6-diamidine-2-phenylindole (DAPI) (Molecular Probes). The cultured cells were examined by a fluorescent microscope (Leica, Wetzlar, Germany) and photographed.
On the other hand, the fresh-frozen SLNs tissues were fixed with 100% acetone at 4°C. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 30 min at room temperature. The tissues were incubated for 1 h at room temperature with primary polyclonal antisera E-selectin (R & D Systems) and ICAM-1 (R & D Systems) and then for 30 min at room temperature with horseradish peroxidase-labeled antirabbit IgG and antimouse IgG (Nichirei, Tokyo, Japan). The reaction product was developed using the DAB kit (Nichirei). The nuclei of the SLN tissues were also counter stained using hematoxylin staining. The SLN tissues were examined by a light microscope (Leica) and photographed.
To examine the effects of chemokines on the immunohistochemical expression of adhesion molecules on the LECs, the starvation culture medium was exchanged for 1 mL of EBM-2 with 3% FBS containing various concentrations of chemokines and then stimulated the human LECs for 4, 18 or 48 h. The various concentrations of chemokines were constructed by diluting each chemokine with appropriate volumes of EBM-2 with 3% FBS.
In some experiments, the effects of 10 ng/mL CCL2 on the immunohistochemical expression of ICAM-1 on the human LECs were also evaluated using the same procedure as above, after overnight neutralization of CCL2 in the starvation culture medium with a CCL2-specific antibody (1.0 µg/mL).
To obtain positive controls of primary antisera to E-selectin, P-selectin, VCAM-1 and ICAM-1, we examined the effects of 18-h stimulation of 10 ng/mL tumor necrosis factor (TNF)-α or 100 ng/mL lipopolysaccharide (LPS) on the immunohistochemical expression of the adhesion molecules on the human LECs.
For non-specific staining, Block-ace (Dainippon Sumitomo Pharma, Osaka, Japan) was substituted for primary antisera as a negative control.
To quantitatively examine the immunohistochemical data concerning the expression of ICAM-1 on the human LECs, the high-resolution digital microphotographs were processed using the Scion image analysis program. The constant area of each of the LECs was outlined on the gray scale image and processed for density measurement. The results were expressed in arbitrary units (mean density per pixel). The data are shown as mean ± SEM (n = 5).