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Uppsala University

18 ARTICLES PUBLISHED IN JoVE

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Biology

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Anna Karlgren 1, Jenny Carlsson 2, Niclas Gyllenstrand 2, Ulf Lagercrantz 1, Jens F. Sundström 2
1Department of Evolutionary Functional Genomics, Evolutionary Biology Center, Uppsala University, 2Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

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Medicine

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease
Jörg Hanrieder 1,2, Anna Ljungdahl 1, Malin Andersson 1
1Department of Pharmaceutical Biosciences, Uppsala University, 2Department of Chemical and Biological Engineering, Chalmers University of Technology

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

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Biology

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival
Per Wessman 1, Sebastian Håkansson 1, Klaus Leifer 2, Stefano Rubino 2
1Department of Microbiology, Uppsala Biocenter, Swedish University of Agricultural Sciences, 2Department of Engineering Sciences, Uppsala University

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

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Biology

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam
Stefano Rubino 1,4, Petter Melin 3, Paul Spellward 2, Klaus Leifer 1
1Department of Engineering Sciences, Uppsala University, 2Gatan Inc., 3Department of Microbiology, Swedish University of Agricultural Sciences, 4Physics Department, University of Oslo

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

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Immunology and Infection

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium
Sofia Nordling 1, Bo Nilsson 1, Peetra U. Magnusson 1
1Department of Immunology, Genetics and Pathology, Uppsala University

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

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Chemistry

Transport of Surface-modified Carbon Nanotubes through a Soil Column
Prabhakar Sharma 1,2, Fritjof Fagerlund 2
1School of Ecology and Environmental Studies, Nalanda University, 2Department of Earth Sciences, Uppsala University

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

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Developmental Biology

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments
Shahrzad Shirazi Fard 1, Maria Blixt 1, Finn Hallböök 1
1Department of Neuroscience, Biomedical Center, Uppsala University

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

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Developmental Biology

Dissection and Culture of Mouse Embryonic Kidney
Bejan Aresh 1, Christiane Peuckert 2
1Department of Neuroscience, Uppsala University, 2Department of Organismal Biology, Evolutionary Biology Center, Uppsala University

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

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Chemistry

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene
Rebecca A. Ryan 1, Sophie Williams 1, Andrew V. Martin 1, Ruben A. Dilanian 1, Connie Darmanin 2, Corey T. Putkunz 1, David Wood 3, Victor A. Streltsov 4, Michael W.M. Jones 5, Naylyn Gaffney 6, Felix Hofmann 7, Garth J. Williams 8, Sebastien Boutet 9, Marc Messerschmidt 10, M. Marvin Seibert 11, Evan K. Curwood 11, Eugeniu Balaur 2, Andrew G. Peele 5, Keith A. Nugent 2, Harry M. Quiney 1, Brian Abbey 2
1ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, 2Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, 3Department of Physics, Imperial College London, 4Florey Institute of Neuroscience and Mental Health, 5Science and Engineering Faculty, Queensland University of Technology, 6Swinburne University of Technology, 7Department of Engineering Science, University of Oxford, 8Brookhaven National Laboratory, 9Linac Coherent Light Source, SLAC National Accelerator Laboratory, 10BioXFEL Science and Technology Center, 11Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, 12Australian Synchrotron

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

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Biochemistry

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)
Xiaoying Qi *1, Yunyun Zhang *2, Yuan Zhang 1, Tianhui Ni 3, Wenfeng Zhang 2, Chunhua Yang 1, Jia Mi 1,4, Jiandi Zhang 2,3, Geng Tian 1
1Medicine and Pharmacy Research Center, Binzhou Medical University, 2Yantai Zestern Biotechnique Co. LTD, 3Precision Medicine research center, Binzhou Medical University, 4Department of Chemistry - BMC, Uppsala University

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

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Environment

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies
Sarah Nicholas 1, Malin Thyselius 2, Marissa Holden 1, Karin Nordström 1,2
1Centre for Neuroscience, Flinders University, 2Department of Neuroscience, Uppsala University

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

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Cancer Research

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation
Andreas Agathangelidis *1, Lesley Ann Sutton *2,3, Anastasia Hadzidimitriou 1, Cristina Tresoldi 4, Anton W. Langerak 5, Chrysoula Belessi 6, Frederic Davi 7, Richard Rosenquist 2,3, Kostas Stamatopoulos 1,2, Paolo Ghia 8
1Institute of Applied Biosciences, Centre for Research and Technology Hellas, 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 3Department of Molecular Medicine and Surgery, Karolinska Institutet, 4Division of Immunology, Transplantation and Infectious, IRCCS San Raffaele Scientific Institute, 5Department of Immunology, Laboratory for Medical Immunology, Erasmus University Medical Center, 6Hematology Department, Nikea General Hospital, 7Assistance publique - Hôpitaux de Paris (AP-HP), Hopital Pitié-Salpêtrière, Department of Hematology, and UPMC University Paris 06, UMRS 1138, 8Division of Experimental Oncology, IRCCS Istituto Scientifico San Raffaele and Università Vita-Salute San Raffaele

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

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Immunology and Infection

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
Zhengkang Luo 1, Lina Thorvaldson 1, Martin Blixt 1, Kailash Singh 1
1Department of Medical Cell Biology, Uppsala University

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

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JoVE Core

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids
Jorik M. van Rijn 1,2,3, Marliek van Hoesel 1,2, Sabine Middendorp 1,2
1Division of Pediatrics, Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, 2Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, 3Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

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Behavior

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse
Zisis Bimpisidis 1, Niclas König 1, Åsa Wallén-Mackenzie 1
1Department of Organismal Biology, Unit of Comparative Physiology, Uppsala University

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

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Cancer Research

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties
Carlemi Calitz 1, Nataša Pavlović 1, Jenny Rosenquist 2, Claudia Zagami 1, Ayan Samanta 2, Femke Heindryckx 1
1Department of Medical Cell Biology, Uppsala University, 2Polymer Chemistry, Department of Chemistry-Ångström Laboratory, Uppsala University

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

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Biochemistry

Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms
Tullia Calogiuri 1,2, Mathilde Hagens 2, Jan Willem Van Groenigen 1, Thomas Corbett 3, Jens Hartmann 4, Rick Hendriksen 5, Iris Janssens 6, Ivan A. Janssens 7, Guillermo Ledesma Dominguez 7, Grant Loescher 4, Steven Mortier 6, Anna Neubeck 3, Harun Niron 7, Reinaldy P. Poetra 4, Lukas Rieder 4, Eric Struyf 7, Michiel Van Tendeloo 8, Tom De Schepper 6, Tim Verdonck 9, Siegfried E. Vlaeminck 8, Sara Vicca 7, Alix Vidal 1
1Soil Biology Group, Wageningen University & Research, 2Soil Chemistry and Chemical Soil Quality, Wageningen University & Research, 3Department of Earth Sciences, Uppsala University, 4Institute for Geology, Center for Earth System Research and Sustainability, University of Hamburg, 5Tupola, Wageningen University & Research, 6IDLab - Department of Computer Science, University of Antwerp - imec, 7Plants and Ecosystems (PLECO), Biology Department, University of Antwerp, 8Research Group of Sustainable Energy, Air and Water Technology, University of Antwerp, 9Department of Mathematics, University of Antwerp - imec

Here we present the construction and operation of an experimental setup to enhance mineral weathering through the activity of soil organisms while concurrently manipulating abiotic variables known to stimulate weathering. Representative results from the functioning of the setup and sample analyses are discussed together with points for improvement.

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Developmental Biology

Z-Scores for Assessing Ovarian Reserve in Young Patients Undergoing Fertility Preservation
Jasmin Hassan 1,2, Pauliina Damdimopoulou 1,2, Atte Lahtinen 3,4, Kirsi Jahnukainen 5,6, Katri Knuus 7, Ganesh Acharya 1, Richelle D. Björvang 1,8,9
1Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 2Department of Gynecology and Reproductive Medicine, Karolinska University Hospital, 3Applied Tumor Genomics Research Program, Faculty of Medicine, University of Helsinki, 4Department of Medical and Clinical Genetics, Medicum, Faculty of Medicine, University of Helsinki, 5Children's Hospital, Pediatric Research Centre, University of Helsinki and Helsinki University Hospital, 6NORDFERTIL Research Lab Stockholm, Department of Women's, and Children's Health, Karolinska Institutet, and Karolinska University Hospital, 7Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, 8Department of Cell and Molecular Biology, Uppsala University, 9Department of Women's and Children's Health, Uppsala University

The present protocol describes a method for assessing ovarian reserve in patients under 25 years old who require fertility preservation through ovarian tissue cryopreservation. This method involves: (1) histological assessment of ovarian reserve in cortical samples, (2) comparison to a reference dataset, and (3) calculation of Z-scores.

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