Goat anti gpnmb

Serial sections of formalin-fixed tissues were deparaffinized, rehydrated, immersed in citrate buffer (pH 6.0), and microwaved for 15 minutes to retrieve antigens. Slides were incubated in 5% donkey, horse, or goat serum overnight at 4°C, stained for 1 hour with goat anti-GPNMB (1:1,000 dilution; R&D Systems), mouse anti-B7-H1/PDL1 (1:500 dilution; R&D Systems), mouse anti-Cytokeratin 20 (1:500 dilution; Invitrogen), rabbit anti-CD3 (1:500 dilution; Novus Bilogicals) or rabbit anti-CD14 (1:500 dilution; Novus Bilogicals). After washing, slides were incubated with biotin-secondary Ab and avidin-peroxidase solution (Vector), followed by color development (brown) by 3,3-diaminobenzidine and counterstaining with hematoxylin. These IHC-stained sections were examined for expression of receptors in a blinded-fashion. Frequency of receptor expression in total CD14⁺ cells was determined as follows: percentage of DC-HIL⁺ CD14⁺ cells was determined by the area of anti-DC-HIL-positive staining relative to that of anti-CD14-staining using image analysis. Data shown are the average of five separate microscopic views.