Normal antibody diluent

Pancreatic cancer tissue slides were obtained from Department of Pathology at Sparrow Hospital, Lansing MI. The use of archived specimens for this study was approved by Michigan State University (MSU) and Sparrow Hospital Institutional Review Boards (IRBs). Our IRB committee waived the need for consent. Nine patients who underwent pancreatic resection for pancreatic ductal adenocarcinoma from 2002 though 2012 were selected and were included in this study. The archived formalin-fixed, paraffin-embedded specimens were sectioned on a rotary microtome at 4 µm’s. Enzyme induced epitope retrieval was performed by 0.03% protease E for 10 minutes at 37°C. Anti-PTK6 antibody was diluted in 1/100 with Normal Antibody Diluent (NAD)(Scytek – Logan, UT) and incubated for 1 hour at room temperature. Antigen-antibody reactions were visualized with the avidin-biotin-peroxidase complex system (R.T.U. Vectastain Elite ABC Reagent; Vector Laboratories, Burlingame, CA, USA). The slides were reviewed and PTK6 expression was graded according to cytoplasmic staining intensity as follows; negative, no staining or weak intensity staining in less than 5% of cells; weak to moderate positive, weak to moderate intensitiy; strong positive, strong intensity.

CAMs were collected from ECEs, washed in PBS, fixed in neutral buffered 10% formalin in PBS for 24 h, stored in 70% ethanol and finally paraffin-embedded. Four micrometer sections of CAM were mounted on glass slides and subsequently deparaffinized and rehydrated in alcohol series. Next, the sections were subjected to endogenous peroxidase inactivation and antigen retrieval as described before [40 ]. Sections were washed in phosphate buffered Normal Antibody Diluent (NAD, ScyTek Laboratories, Logan, USA) containing 0.1% Tween-20, and after primary antibody incubation with PBS 0.1% Tween-20. Sections were incubated for 60 min at room temperature with MAb Ch/IBV 26.1 diluted 1:100 in NAD. Antibody binding was detected by Dako Envision HRPO labeled polymer anti-mouse (Dako, by Agilent Technologies, Santa Clara, USA) diluted 1:1 in NAD, and visualized by 3-Amino-9-ethylcarbazole (AEC, Dako). Slides were counterstained with hematoxylin, mounted with Aquatex (Merck, Darmstadt, Germany), and viral antigen presence was assessed by light microscopy (BX60, Olympus, Tokyo, Japan).