Clone ov tl 12 30

Five µm sections of paraffin-embedded tissues and PDOs were stained with haematoxylin and eosin (H&E) and immunohistochemistry staining was performed with appropriate primary monoclonal rabbit anti-human antibodies: WT1 (clone 6F-H2, Roche Diagnostics, Indianapolis, IN), PAX8 (PAX8-EP331), p53 (clone Bp53-11 Roche), anti-CINtec ® Histology Kit (p16) (Roche) using the UltraView Universal DAB Detection Kit on the ULTRA instrument (Ventana) BenchMark. For cytokeratin 7 (CK7; clone OV-TL 12/30 DAKO/AGILENT) and CK20 (Clone Ks20.8 DAKO/AGILENT) staining was performed on the Bond III automated immunostainer (Leica Microsystems, Bannockburn, IL). Appropriate positive and negative controls were included. The selected antibodies are well-established in the diagnostic routine laboratory, signals were clearly visible and captured by ordinary light microscope (Zeiss AxioPhot Microscope). Scores were performed by an expert pathologist.

Tumor tissue specimens were formalin-fixed and paraffin-embedded, sectioned (4 μm) and stained with hematoxylin and eosin. Immunohistochemical analyses were performed with a primary polyclonal mouse anti-human antibody directed against trypsin (1:2,000; Qed Bioscience Inc., San Diego, CA, USA), monoclonal mouse anti-human antibodies directed against cytokeratin 7 (1:50; clone OV-TL 12/30; DakoCytomation, Glostrup, Denmark), cytokeratin 18 (1:10; clone DC10; DakoCytomation), synaptophysin (1:2; clone Snp 88; BioGenex, San Ramon, CA, USA), chromogranin A (1:2; clone LK2H10; Linearis Beratungs-GmbH, Wertheim, Germany), Ki-67 (1:100; DakoCytomation), p53 (1:100; clone DO7; DakoCytomation), β-catenin (1:200; clone 14; BD Transduction Laboratories, Lexington, KY, USA) and epidermal growth factor receptor (1:50; clone 31G7; Zymed Laboratories Inc., San Francisco, CA, USA), as well as a polyclonal rabbit anti-human antibody directed against Smad4 (1:50; rabbit polyclonal; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using the avidin-biotin complex method. If necessary, antigen retrieval in the sections was achieved by microwave pretreatment in citrate buffer (used for trypsin, p53, Smad4 and β-catenin).

Collection of porcine pancreatic tissue and experimental procedures were conducted in accordance with the institutional ethical committee of the University of Alberta and the Canadian Council of Animal Care. Briefly, donor pancreata were surgically removed from either sex neonatal piglets (Swine Research and Technology Center, University of Alberta, Edmonton AB CAN). Isolation and culture of neonatal porcine islets were performed as characterized previously^{57 (link)}. Islets were treated with 10 μM folinic for 5 days. Then, the islets were either fixed, sectioned, and stained for insulin (1:5; DAKO, code# IR002), glucagon (1:5000; Sigma-Aldrich), somatostatin (1:300; DAKO, code# A0566), and CK7 (3:100; DAKO, clone OV/TL 12/30) or further processed for GSIS analysis as previously described^{58 (link)}.

The 4 μm-thick, FFPE slices were de-paraffinized and rehydrated using a xylene and alcohol solution. Immunostaining was performed using automated instruments (Ventana Benchmark XT (Ventana Medical Systems) and or Dako Omnis (Dako, Carpinteria, CA, USA)) [1 (link),18 (link),19 (link),20 (link),21 (link),22 (link),23 (link),24 (link),25 (link),26 (link)]. After antigen retrieval, the slices were incubated with primary antibodies including pan-cytokeratin (pan-CK; 1:600, clone AE1/AE3, Dako), CK7 (Dako, 1:100, clone OV-TL 12/30, Dako), CK20 (1:100, clone Ks20.8, Dako), caudal type homeobox 2 (CDX2; 1:400, clone EPR2764Y, Cell Marque, Rocklin, CA, USA), paired box 8 (PAX8; 1:50, polyclonal, Cell Marque), estrogen receptor (ER; 1:150, clone 6F11, Novocastra, Leica Biosystems, Newcastle Upon Tyne, UK), progesterone receptor (PR; 1:100, clone 16, Novocastra), p53 (1:300, clone DO-7, Novocastra), p16 (prediluted, clone E6H4, Ventana Medical Systems), p63 (1:50, clone 4A4, Dako), and GATA-binding protein 3 (GATA3; 1:150, clone L50-823, Cell Marque). After chromogenic visualization, the slices were counterstained with hematoxylin. Appropriate positive and negative controls were concurrently stained to validate the staining method [27 (link),28 (link),29 (link)]. Negative controls were prepared by substituting non-immune serum for primary antibodies, resulting in no detectable staining.

Immunohistochemical staining for HER2 was performed using the HercepTest™ kit (Dako, Glostrup, Denmark) according to manufacturer's protocols. 4 μm thick sections taken both from original full tumor blocks and the TMA block were used for HER2 IHC. HER2 protein expression was scored as 0, 1+, 2+, and 3+ according to the ASCO/CAP 2013 HER2 test guideline [26 (link)]. HER2 IHC 3+ was considered to be HER2-positive, IHC 2+ HER2-equivocal, and IHC 0 and 1+ HER2-negative [26 (link)]. Additional immunohistochemical staining for cytokeratin (CK) 7 (clone OV-TL 12/30; Dako), CK20 (clone Ks20.8; Dako), p53 (clone DO7; Dako), p63 (clone 4A4; Ventana), and CD138 (clone 5F7; Novocastra) was also performed on sections taken from the TMA block. All IHC were performed using Ventana Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ).