Primary monoclonal antibodies

Mice were sacrificed on days 7, 15 and 23 after melanoma inoculation. The most common metastatic organs (liver, spleen, kidney, adrenal glands, liver and lungs) were resected, sectioned, and fixed by immersion in 4% formaldehyde for 24 h. Organs were subsequently embedded in paraffin and cut at 3-μm thickness for histological analysis. Different sections (stained with hematoxylin-eosin) of each organ were analysed by two independent pathologists. For immunohistochemical analysis of lymphocyte markers, EnVision technology (Dako) was used. Samples were boiled in target retrieval solution buffer (pH 6 for CD23, pH 8 for the rest) for 20 min, subsequently cooled in distilled water and 1×PBS. Next, the sections were incubated with ready-to-use primary monoclonal antibodies (Dako) against CD4 (clone 4B12), CD8 (clone c8/144B), CD23 (clone DAK-CD23), CD45 (clone 2B11 + PD7/26), CD56 (clone 123C3) and CD68 (clone KP1). The antigens were visualized using biotinylated antibodies and streptavidin conjugated with horseradish peroxidase (EnVision Mouse HRP, Dako). Diaminobenzidine (DAB, Dako) was used as the chromogen. Antigen concentrations were all determined based on the percentage and intensity of tumour cells showing positive staining.

NV mice were sacrificed on days 0, 7 and 14 after B16OVA melanoma transplantation. The most common metastatic organs (liver, spleen, kidney, adrenal glands, liver and lungs) were resected, sectioned, and fixed by immersion in 4% formaldehyde for 24 h. Organs were subsequently embedded in paraffin, processed, and sections stained with hematoxylin–eosin) and immunohistochemical analysis of lymphocyte markers performed as previously described [10 (link)]. Primary monoclonal antibodies (Dako, Carpinteria, CA, USA) used were against the following antigens, CD4 (clone 4B12), CD8 (clone c8/144B), CD23 (clone DAK-CD23), CD45 (clone 2B11 + PD7/26), CD56 (clone 123C3) and CD68 (clone KP1) and visualized as described [10 (link)].

The tumors formed in nude mice were fixed with 10% neutral-buffered formalin and routinely embedded in paraffin wax for histological examination. Sections were stained with hematoxylin and eosin. Serial sections were immunostained using the streptavidin-biotin-peroxidase method with primary monoclonal antibodies specific for Ki67 (1:100, Dako, Denmark A/S, Glostrup, Denmark) and alpha-smooth muscle actin (SMA, 1:400, Dako), vascular endothelial growth factor (VEGF, 1:100, Santa Cruz Biotechnology, California, USA). Briefly, sections were treated in 0.03% H₂O₂ in 33% methanol at room temperature for 30 min for endogenous peroxidase blocking, following a pretreatment at 121°C for 20 min in citrate buffer (pH 6.0) for Ki67 and SMA, and at 121°C for 15min in citrate buffer (pH 9.0) for VEGF. The validation of antibodies was confirmed by a positive reaction with biopsy samples diagnosed with canine mammary adenocarcinoma or by a negative normal mouse IgG. The intratumor SMA-positive vessel and Ki67 index of tumor cell densities were evaluated as previously described (34 (link)). To evaluate the immunostaining intensity of VEGF, 5 high-power field (x400) of tumor tissue were selected and measured using Image J software.