University of Pisa View Institution's Website 11 articles published in JoVE Neuroscience Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging Danila Di Meo*1, Josephine Ramazzotti*1, Marina Scardigli*1,2, Franco Cheli1, Luca Pesce1,3, Niamh Brady1, Giacomo Mazzamuto1,4,5, Irene Costantini1,4,6, Francesco S. Pavone1,4,5 1European Laboratory for Non-linear Spectroscopy (LENS), University of Florence, 2Division of Physiology, Department of Experimental and Clinical Medicine, University of Florence, 3Department of Physics, University of Pisa, 4National Research Council - National Institute of Optics (CNR-INO), 5Department of Physics and Astronomy, University of Florence, 6Department of Biology, University of Florence The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol. Genetics Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes Aurora Falaschi1, Anna Chiaramonte1,2, Serena Testi1, Roberto Scarpato1 1Unità di Genetica, Dipartimento di Biologia, University of Pisa, 2 This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes. Cancer Research Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing Alice Usai1, Gregorio Di Franco2, Chiara Gabellini1, Luca Morelli2, Vittoria Raffa1 1Department of Biology, University of Pisa, 2General Surgery Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue. Medicine Robot-Assisted Radical Antegrade Modular Pancreatosplenectomy Including Resection and Reconstruction of the Spleno-Mesenteric Junction Niccolò Napoli1, Emanuele F. Kauffmann1, Francesca Menonna1, Sara Iacopi1, Concetta Cacace1, Ugo Boggi1 1Division of General and Transplant Surgery, University of Pisa The robotic technique shown herein aims at faithfully reproducing the open procedure for radical treatment of cancer of the body-tail of the pancreas. The protocol also demonstrates the ability to master involvement of major peripancreatic vessels without conversion to open surgery. Neuroscience Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation Maria Paola Tramonti Fantozzi*1, Tommaso Banfi*2,3, Vincenzo De Cicco*1, Massimo Barresi4, Enrico Cataldo5, Davide De Cicco1, Luca Bruschini6, Paola d'Ascanio1, Gastone Ciuti2,3, Ugo Faraguna1,7, Diego Manzoni1 1Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, 2 To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected. Medicine Ultrasound-based Pulse Wave Velocity Evaluation in Mice Nicole Di Lascio1,2, Claudia Kusmic2, Francesco Stea2,3, Francesco Faita2 1 Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites. Cancer Research Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models Rocco Sciarrillo1,2,3, Anna Wojtuszkiewicz1, Irsan E. Kooi4, Valentina E. Gómez3, Ugo Boggi5, Gerrit Jansen6, Gert-Jan Kaspers1,7, Jacqueline Cloos*1, Elisa Giovannetti*3,8,9 1Department of Pediatric Oncology/Hematology, VU University Medical Center, 2Department of Hematology, VU University Medical Center, 3Department of Medical Oncology, VU University Medical Center, 4Department of Clinical Genetics, VU University Medical Center, 5Division of General and Transplant Surgery, Azienda Ospedaliera Universitaria Pisana, Universita’ di Pisa, 6Amsterdam Immunology and Rheumatology Center, VU University Medical Center, 7Princess Máxima Center for Pediatric Oncology, 8Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa, 9Institute of Nanoscience and Nanotechnology, CNR-Nano Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. Bioengineering Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications Luisa Trombi1,2, Serena Danti2,3, Sara Savelli4, Stefania Moscato1, Delfo D'Alessandro2,5, Claudio Ricci2, Stefano Giannotti6, Mario Petrini1 1Dept. of Clinical and Experimental Medicine, University of Pisa, 2OtoLab, Azienda Ospedaliero-Universitaria Pisana (AOUP), 3Dept. of Civil and Industrial Engineering, University of Pisa, 4Immunohematology Operative Unit, Azienda Ospedaliero-Universitaria Pisana (AOUP), 5Dept. Of Surgical, Medical, Molecular Pathology and Emergency Medicine, University of Pisa, 6II Orthopedic and Traumatologic Clinic, Azienda Ospedaliero-Universitaria Pisana (AOUP) Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation. Developmental Biology Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow Marina Montali*1, Serena Barachini*1, Simone Pacini1, Francesca M. Panvini1, Mario Petrini1 1Department of Clinical and Experimental Medicine, University of Pisa Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential. Immunology and Infection Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry Olimpia Gamucci1, Alice Bertero1,2, Maria Ada Malvindi3, Stefania Sabella3, Pier Paolo Pompa3, Barbara Mazzolai1, Giuseppe Bardi1 1Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia, 2Department of Biology, University of Pisa, 3Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry. Medicine Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens Dorothée Sturm1,2, Lorella Marselli3, Florian Ehehalt1,2, Daniela Richter1, Marius Distler2, Stephan Kersting1,2, Robert Grützmann2, Krister Bokvist4, Philippe Froguel5, Robin Liechti6, Anne Jörns7, Paolo Meda8, Gustavo Bruno Baretton9, Hans-Detlev Saeger2, Anke M. Schulte10, Piero Marchetti3, Michele Solimena1 1Molecular Diabetology, Paul Langerhans Institute Dresden, 2Department of GI-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 3Department of Endocrinology and Metabolism, Metabolic Unit University of Pisa, 4Labs DC0522, Lilly Corporate Center, 5Genomics, Faculty of Medicine Imperial College London, 6Vital-IT, SIB Swiss Institute of Bioinformatics, 7Clinical Biochemistry, Hannover Medical School, 8Cell Physiology and Metabolism, Medical School, University of Geneva, 9Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden, 10R&D DIAB Division / Translational Medicine, Sanofi-Aventis Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.