Streptavidin alkaline phosphatase

For immunohistochemistry, the slides were incubated with mouse anti-human HAM56 (Dako Cytomation, Carpinteria, CA), mouse anti-human CD3 (BDBiosciences, San Jose, CA), mouse anti-human CD4 (Abcam, Cambridge, MA), and mouse anti-human CD8 (Abcam, Cambridge, MA) for 1.5 h at 25° C. Primary antibodies were localized with appropriate biotinylated secondary antibodies, streptavidin–alkaline phosphatase (Biogenex, San Ramon, CA) and Vector Red (Vector Labs, Burlingame, CA) substrate. Sections were counterstained with Mayer's Hematoxylin and examined by the study pathologist (JAC) using light microscopy. Thymus and lymph node were used as tissue controls; as an assay control, the primary antibodies were substituted with non-immune sera. Cell densities for macrophages, CD3+ cells, CD4+ cells, and CD8+ cells were determined by overlay of a 25 x 25 pixel grid with evaluation of each crosshatch for positive or negative staining by the pathologist (JAC), who was blinded to treatment. Cell densities are expressed as the percentage of intima occupied by positive staining.

Formalin-fixed tumors were dehydrated and embedded in paraffin. Subsequently, 3-μm tumor sections were prepared and placed on a microscope slide. Prior to incubation with primary antibody, paraffin sections were dewaxed in xylene and rehydrated in ethanol/water. For antigen retrieval, the tumor sections were treated at 99 °C for 25 min in citrate buffer (Target Retrieval Solution, pH 6.0, DAKO, Glostrup, Denmark). The following primary antibody was used: rabbit anti-cleaved caspase 3 (BD Biosciences). The tumor sections were incubated with the primary rabbit antibody diluted 1 : 100 in blocking buffer (PBS+20 mg/ml BSA+1 mg/ml human IgG) for 60 min at room temperature. After a PBS washing step, specific binding of the primary antibody was visualized using an anti-rabbit biotinylated secondary antibody (Southern Biotech, Birmingham, AL, USA) and streptavidin alkaline phosphatase (BioGenex, Fremont, CA, USA). The FAST-Red substrate system (DAKO) was used as the substrate for the alkaline phosphatase, which produced a red precipitate at antibody-binding sites. Sections were then counterstained with Mayer’s hematoxylin and mounted with glycerin-gelatin. A rabbit isotype control antibody was used for control staining.