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Université Libre de Bruxelles

4 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes
Soizic Garaud *1,2, Chunyan Gu-Trantien *1,2, Jean-Nicolas Lodewyckx 1,2, Anaïs Boisson 1,2, Pushpamali De Silva 1,2, Laurence Buisseret 1,2, Edoardo Migliori 1,2, Myriam Libin 3, Céline Naveaux 1,2, Hugues Duvillier 1,4, Karen Willard-Gallo 1,2
1Molecular Immunology Unit, Université Libre de Bruxelles, 2Institut Jules Bordet, Université Libre de Bruxelles, 3Institut d'Immunologie Médicale, Université Libre de Bruxelles, 4Flow Cytometry Core Facility, Université Libre de Bruxelles

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

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Biology

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry
Abdulkadir Abakir 1, Lee Wheldon 2, Andrew D. Johnson 3, Patrick Laurent 1, Alexey Ruzov 4
1Laboratoire de Neurophysiologie (CP601), ULB Neuroscience Institute (UNI), Université Libre de Bruxelles, 2Medical Molecular Sciences, Centre for Biomolecular Sciences, University of Nottingham, 3School of Life Sciences, University of Nottingham, 4Division of Cancer and Stem Cells, Centre for Biomolecular Sciences, School of Medicine, University of Nottingham

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

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Genetics

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images
Ashley H. Ramsawhook *1, Lara C. Lewis *1, Maria Eleftheriou 1, Abdulkadir Abakir 1, Paulina Durczak 1, Robert Markus 2, Seema Rajani 2, Nicholas R.F. Hannan 1, Beth Coyle 3, Alexey Ruzov 1
1Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences, University of Nottingham, 2School of Life Sciences Imaging (SLIM), School of Life Sciences, University of Nottingham, 3Children's Brain Tumour Research Centre, School of Medicine, QMC, University of Nottingham

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

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Biochemistry

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Raphael Dutoit 1,2, Nathalie Brandt 2, Dany Van Elder 1, Louis Droogmans 1
1Laboratory of Microbiology, Department of Molecular Biology, Université Libre de Bruxelles, 2Labiris Institut de Recherche

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

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