Horseradish peroxidase conjugated goat anti rabbit secondary antibody sc 2004

Tissue microarray system (Quick-Ray, UT06,) was used for 2.0-mm tissue in each patient in the TMA analysis. TMA sections were dewaxed with a 100% xylene solution and rehydrated with ethanol gradient solution. Antigen retrieval was carried out by boiling the samples in citrate buffer solution (pH 6.0) for 5 minutes, followed by a 15-minute incubation with PBS containing 5% goat serum to block non-specific binding. TMA sections were incubated with the primary antibody against CXCL2 (PeproTech, Rocky Hill, CT), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The staining of CXCL2 was independently evaluated by 2 pathologists under double-blind conditions. The positive rate of staining was 0% to 100%. The grade of staining intensity was set as follows: 0=no staining; 1 = mild; 2 = moderate; and 3 = high. The final staining score was obtained by multiplying the intensity grade with the proportion of positive cells. The cutoff value expressed by CXCL2 was calculated by the X-tile software program. The degree of staining was quantified by 2 grades, and the final CXCL2 staining score was defined as follows: 0 to 150, low expression; 150 to 300, high expression.