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5 ARTICLES PUBLISHED IN JoVE

Biology

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Biology

Purification and Refolding to Amyloid Fibrils of (His)₆-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Qiaojing Li¹, Charles-Adrien Richard¹, Mohammed Moudjou¹, Jasmina Vidic¹

¹Virologie et Immunologie Moléculaires, INRA

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

JoVE Journal

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Federica Franciosi¹, Irene Tessaro^1,2, Rozenn Dalbies-Tran³, Cecile Douet³, Fabrice Reigner⁴, Stefan Deleuze⁵, Pascal Papillier³, Ileana Miclea⁶, Valentina Lodde¹, Alberto M. Luciano¹, Ghylene Goudet³

¹Department of Health, Animal Science and Food Safety, University of Milan, ²IRCCS. Istituto Ortopedico Galeazzi, ³PRC, CNRS, IFCE, Université de Tours, INRA, ⁴PAO, INRA, ⁵Clinique des Animaux de Compagnie et des Équidés, Université de Liège, ⁶University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

Biochemistry

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Javier A. Jaimes^*1,2, Jean K. Millet^*1,3, Monty E. Goldstein^1,4, Gary R. Whittaker¹, Marco R. Straus¹

¹Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, ²Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, ³Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA, ⁴Department of Cell Biology and Molecular Genetics, University of Maryland

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Immunology and Infection

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

Jean K. Millet^1,2, Tiffany Tang³, Lakshmi Nathan³, Javier A. Jaimes⁴, Hung-Lun Hsu^3,5, Susan Daniel³, Gary R. Whittaker¹

¹Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, ²INRA, Virologie et Immunologie Moléculaires, ³Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, ⁴Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, ⁵Horae Gene Therapy Center, University of Massachusetts Medical School

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.