br Immunofluorescence staining br Pancreatic
Pancreatic cancer tissue from KPf/fC mice was Þxed in Z-Þx (Anatech Ltd, Fisher ScientiÞc) and parafÞn embedded at the UCSD His-tology and Immunohistochemistry Core at The Sanford Consortium for Regenerative Medicine according to standard protocols. 5 mm sections were obtained and deparafÞnized in xylene. The human pancreas parafÞn embedded tissue array was acquired from US Biomax, Inc (BIC14011a). For parafÞn embedded mouse and human pancreas tissues, antigen retrieval was performed for 40 minutes in 95-100 C 1x Citrate Buffer, pH 6.0 (eBioscience). Sections were blocked in PBS containing 0.1% Triton X-100 (Sigma- Aldrich), 10% Goat Serum (Fisher ScientiÞc), and 5% bovine serum albumin (Invitrogen).
KPf/fC cells and human pancreatic cancer cell lines were suspended in DMEM (GIBCO, Life Technologies) supplemented with 50% FBS and adhered to slides by centrifugation at 500 rpm. 24 hours later, cells were Þxed with Z-Þx (Anatech Ltd, Fisher ScientiÞc), washed in PBS, and blocked with PBS containing 0.1% Triton X-100 (Sigma-Aldrich), 10% Goat serum (Fisher ScientiÞc), and 5% bovine serum albumin (Invitrogen). All incubations with primary PKF118-310 were carried out overnight at 4 C. Incubation with Alexaßuor-conjugated secondary antibodies (Molecular Probes) was performed for 1 hour at room temperature. DAPI (Molecular Probes) was used to detect DNA and images were obtained with a Confocal Leica TCS SP5 II (Leica Microsystems). The following primary antibodies were used: chicken anti-GFP (Abcam, ab13970) 1:500, rabbit anti-RORg (Thermo Fisher, PA5-23148) 1:500, mouse anti-E-Cadherin (BD Biosciences, 610181) 1:500, anti-Keratin (Abcam, ab8068) 1:15, anti-Hmga2 (Abcam. Ab52039) 1:100, anti-Celsr1 (EMD Millipore abt119) 1:1000, anti-Celsr2 (BosterBio A06880) 1:250.
9.5-10.5 week old REM2-KPf/fC mice were treated either vehicle or SR2211 (10 mg/kg i.p., daily) for 8 days. For imaging, mice were anesthetized by intraperitoneal injection of ketamine and xylazine (100/20 mg/kg). In order to visualize blood vessels and nuclei, mice were injected retro-orbitally with AlexaFluor 647 anti-mouse CD144 (VE-cadherin) antibody and Hoechst 33342 immediately following anesthesia induction. After 25 minutes, pancreatic tumors were removed and placed in HBSS containing 5% FBS and 2mM EDTA. 80-150 mm images in 1024 3 1024 format were acquired with an HCX APO L20x objective on an upright Leica SP5 confocal system using Leica LAS AF 1.8.2 software. GFP cluster sizes were measure using ImageJ 1.51 s software. 2 mice per treat-ment group were analyzed in this study; 6-10 frames were analyzed per mouse.
Analysis of tissue microarrays
Immunohistochemistry (IHC) and staining analysis
TMAs were sectioned to 2.5 mm thickness. IHC staining was performed on a Leica BOND RX automated immunostainer using BOND primary antibody diluent and BOND Polymer ReÞne DAB Detection kit according to the manufacturerÕs instructions (Leica Bio-systems). Pre-treatment was performed using citrate buffer at 100 C for 30 min, and tissue was stained using rabbit anti-human RORg(t) (polyclonal, #PA5-23148, Thermo Fisher ScientiÞc) at a dilution of 1:4000. Stained slides were scanned using a Pannoramic P250 digital slide scanner (3DHistech). RORg(t) staining of individual TMA spots was analyzed in an independent and randomized manner by two board-certiÞed surgical pathologists (C.M.S and M.W.) using Scorenado, a custom-made online digital TMA analysis tool. Interpretation of staining results was in accordance with the ÔÔreporting recommendations for tumor marker prognostic studiesÕÕ (REMARK) guidelines. Equivocal and discordant cases were re-analyzed jointly to reach a consensus. RORg(t) staining in tumor cells was classiÞed microscopically as 0 (absence of any cytoplasmic or nuclear staining), 1+ (cytoplasmic staining only), and 2+ (cyto-plasmic and nuclear staining). For patients in whom multiple different scores were reported, only the highest score was used for further analysis. Spots/patients with no interpretable tissue (less than 10 intact, unequivocally identiÞable tumor cells) or other arti-facts were excluded.