Immunohistochemistry for hTERT

Immunohistochemistry for hTERT was performed using Histostain-SP Detection Kit (Zymed Laboratories, San Francisco, CA). For peroxidase quenching, the sections were incubated at room temperature, for 15 min, in 3% hydrogen peroxide and methanol, and then blocked with blocking solution (reagent A from the above commercial kit) for 15 min and washed twice with PBS. The slides were incubated at room temperature for 1 h with rabbit monoclonal antibody against hTERT (Novus Biologicals, Littleton, CO) at a dilution of 1:50 and then for 10 min with secondary antibody (Reagent B, biotinylated secondary antibody). The slides were then incubated at room temperature for 10 min with streptavidin and washed twice with PBS. The nuclei were stained with DAB for 3 min and washed with distilled water. The slides were then stained with hematoxylin and eosin, washed with water, covered, and viewed with a light microscope. hTERT expression was evaluated based on presence of the signal. We counted 100 cells per slide and grouped cells according to presence or absence of staining for hTERT. The slides were scored blindly, twice.