Envision hrp reagent

The EnVision two-step method was used for immunohistochemistry staining, and the experimental procedure was carried out according to the kit instructions. The tissue slides were dewaxed by xylene, hydrated by gradient ethanol (100-30%), rinsed with distilled water for 3 min, and then placed in 0.01 mol/L sodium citrate buffer (pH 6.0) and microwaved for 5 min for antigen retrieval, and naturally cooled at room temperature. The slides were washed with 0.01 mol/L PBS (pH 7.4) for three times, and subjected to Dual Endogenous Enzyme Block for 10 min. After washing in 0.01 M PBS (pH 7.4) for three times, the slides were incubated overnight with the diluted primary antibody or the PBS solution as the negative control. After washing with 0.01 mol/L PBS (pH 7.4) for three times, the slides were subjected to 30 min DakoCytomation EnVision+ HRP reagent incubation for anti-mouse and anti-rabbit secondary antibodies, or 15 min Biotinylated Link coupled with 15 min Streptavidin-HRP incubation for goat antibodies. Signals were detected by adding substrate hydrogen peroxide using diaminobenzidine as a chromogen and followed by 2 min hematoxylin counterstaining. In the end, all the slides were routinely dehydrated, transparence, sealed and observed under Nikon Eclipse 600 Microscope (Nikon, Tokyo, Japan).

All the PDAC and normal tissues were collected from the Department of Hepatobiliary Surgery, The first Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China. The Collection and use of clinical samples was approved by the Ethical Committee of Hospital and informed consent was obtained from all patients. The tissue sections were deparaffinized in 60˚C for 4h and immersed in xylene quickly, rehydrated in a series of grade alcohols. After washing 3 times by 5 minutes once with PBS buffer (exclusive of potassium), tissue sections were subjected to 5-min pressure cooker antigen retrieval methods, 10-min of quenching endogenous enzyme. Next, the primary antibody was incubated at 4˚C overnight, and Dako Cytomation EnVision-HRP reagent was incubated for 30 mins at room temperature after washing with PBS. Then, diaminobenzidine (DAB) was added to detect the signals and hematoxylin to stain nucleus. The intensity of staining was evaluated by image J software (NIMH, National Institutes of Health, USA).