3 3 diaminobenzidine dab

Paraffin sections for immunohistochemical staining were placed on poly-L-lysine-coated slide (Fisher scientific, Pittsburgh, PA, USA). Slides were immunostained by Invitrogen's HISOTO-STAIN-SP kits using the Labeled-(strept) Avidin-Biotin (LAB-SA) method. After antigen retrieval, slides were immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity and rinsed with PBS. After being rinsed, slides were incubated with 10% nonimmune goat serum for 10 min at room temperature and incubated with primary antibodies of ICAM-1, VCAM-1, and E-selectin (1:200; Santa Cruz, CA, USA) in humidified chambers overnight at 4°C. All slides were then incubated with biotinylated secondary antibody for 20 min at room temperature and then incubated with horseradish peroxidase-conjugated streptavidin for 20 min at room temperature. Peroxidase activity was visualized by 3,3′-Diaminobenzidine (DAB; Novex, CA) substrate-chromogen system, counterstaining with hematoxylin (Zymed, CA, USA). For quantitative analysis, the average score of 10~20 randomly selected area was calculated by using NIH Image analysis software, Image J (NIH, Bethesda, MD, USA).

Parraffin sections for immunohistochemical staining were placed on poly-l-lysine-coated slides (Fisher Scientific, Pittsburgh, PA, USA). Slides were immunostained by Invitrogen’s HISOTO-STAIN^®-SP kits (Carlsbad, CA, USA) using the Labeled-[strept] Avidin-Biotin (LAB-SA) method. After antigen retrieval, slides were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. After being rinsed with PBS, slides were incubated with 10% non-immune goat serum for 10 min and incubated with primary antibodies of ICAM-1, VCAM-1 and ET-1 (1:200; Santa Cruz, CA, USA) in humidified chambers overnight at 4 °C. All slides were then incubated with biotinylated secondary antibody for 20 min, and then incubated with horseradish peroxidase-conjugated streptavidin for 20 min. Peroxidase activity was visualized by 3,3′-Diaminobenzidine (DAB; Novex^®, Los Angeles, CA, USA) substrate-chromogen system, and counterstaining with hematoxylin (Zymed, Carlsbad, CA, USA). For quantitative analysis, the average scores of 10–20 randomly selected areas were calculated by using NIH Image analysis software, Image J (NIH, Bethesda, MD, USA).

Prior to immunohistochemical staining, tissue sections in paraffin were mounted on poly-l-lysine-coated slides (Fisher Scientific, Pittsburgh, PA, USA). The tissue on each slide was immunostained using Invitrogen HISOTO-STAIN-SP kits with the labeled streptavidin-biotin (LAB-SA) method. After antigen retrieval, slides were immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity and rinsed with PBS. Next, slides were incubated with 10% nonimmune goat serum for 10 min at room temperature and incubated with primary antibodies against ICAM-1, VCAM-1, ET-1, and eNOS (1 : 200; Santa Cruz, CA, USA) in humidified chambers overnight at 4°C. Next, slides were incubated with biotinylated secondary antibodies for 20 min at room temperature, followed by incubation with horseradish peroxidase-conjugated streptavidin for 20 min at room temperature. Peroxidase activity was visualized using a 3,3′-diaminobenzidine (DAB; Novex, CA) substrate-chromogen system with hematoxylin counterstaining (Zymed, CA, USA). For quantitative analysis, the average score of 10–20 randomly selected areas was calculated using NIH Image Analysis Software, ImageJ (NIH, Bethesda, MD, USA).