Antibody diluent and background reducing component

The use of the patient samples was approved by Human Ethical Committee of Xiangya Hospital, Central South University. For detection of LMP1 and TAZ, consecutive slide-mounted NPC and gastric cancer sections were first treated with proteinase K at room temperature for 15 min. Endogenous peroxidase activity was inhibited by incubating with 3% H₂O₂ (DAKO, Carpinteria, CA, USA). Nonspecific binding was blocked with Antibody Diluent and Background Reducing Component (DAKO, Carpinteria, CA, USA). Sections were then incubated with anti-TAZ (CST, Danvers, MA, USA; 1:50 dilution) and anti-LMP1 (DAKO, Carpinteria, CA, USA; 1:100 dilution) antibodies at room temperature for 1 h. After a washing step, a HRP-conjugated secondary antibody was added and sections were incubated at room temperature for 20 min. Tissue sections were then treated with DAB reagent (DAKO, Carpinteria, CA, USA); 3, 3′-Diaminobenzidine tetrahydrochloride was used as a chromogen. All images were acquired on an Olympus BX51 microscope (Leica, Germany).

Slide-mounted tissue sections were treated with proteinase K at room temperature for 15 min and incubated with 3% H₂O₂ (DAKO) to inhibit endogenous peroxidase activity. Nonspecific binding was blocked with Antibody Diluent and Background Reducing Component (DAKO). The sections were then incubated with an anti-CDH1 (E-cadherin, Cell signaling #4065; 1∶50 dilution), anti-GFP (GeneTex, 1∶50 dilution) or anti-Mac-2BP antibody (a gift from Dr. Jau-Song Yu, Chung Gung University) at room temperature for 1 hr. After washing, the slides were incubated with an HRP-conjugated secondary antibody at room temperature for 20 min and developed with 3,39-diaminobenzidine tetrahydrochloride (DAB) as a chromogen. All images were acquired on an Olympus BX51 microscope (Olympus, Japan).