Horseradish peroxidase conjugated avidin

Formalin-fixed paraffin-embedded samples were sectioned into 4 μm slices. Deparaffinized sections were subjected to antigen retrieval with citrate solution, pH 6.0 and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After blocking slides with fetal bovine serum, sections were incubated overnight at 4 °C with a 1:100 dilution of CD4 monoclonal antibody (Abcam) and CD8 monoclonal antibody (Santa Cruz Biotechnology). Slides were treated with biotin-conjugated secondary anti-mouse antibody (Dako) and with horseradish peroxidase–conjugated avidin (Dako). Peroxidase activity was localized for all samples with 3,3′-diaminobenzidine, counterstained with Harrys’s hematoxylin, dehydrated, cleared and mounted in mounting medium (Entellan, Merck).

Formalin-fixed paraffin-embedded samples were sectioned into 4-μm slices. Deparaffinized sections were subjected to antigen retrieval with citrate solution, pH 6.0, and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After blocking slides with FBS, sections were incubated overnight at 4°C with a 1:200 dilution of anti-vascular endothelial growth factor (VEGF) (Abcam, ab1316), 1:100 dilution of anti-VCAM-1 (Santa Cruz Biotechnology, sc-1504), anti-CD4 (Abcam, ab51312), and anti-CD8 (Santa Cruz Biotechnology, sc-7970) monoclonal antibody. Slides were treated with biotin-conjugated secondary antibody (Dako) and with horseradish peroxidase-conjugated avidin (Dako). Peroxidase activity was localized for all samples with 3,3′-diaminobenzidine, counterstained with hematoxylin, dehydrated, cleared, and mounted in mounting medium Entellan (Merck).