Journal of Visualized Experiments

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Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Launch Video Published: 02/22/2012 ID: 3668
Daniel Anacker1
Cary Moody1, 2

1Department of Microbiology & Immunology, University of North Carolina-Chapel Hill

2Lineberger Cancer Center, University of North Carolina-Chapel Hill

Immunology, Issue 60, Epithelium, organotypic raft culture, virus, keratinocytes, papillomavirus,
Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Launch Video Published: 02/22/2012 ID: 3606
Andrew R. Bauder
Toby A. Ferguson

Center for Neural Repair and Rehabilitation, Temple University

Neuroscience, Issue 60, Sciatic nerve crush, regeneration, neuromuscular junction, muscle whole mount, mouse,
Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Launch Video Published: 02/21/2012 ID: 3894
Toni G. Patton1
Gabrielle Dietrich2
Kevin Brandt1
Marc C. Dolan2
Joseph Piesman2
Robert D. Gilmore Jr.1

1Microbiology and Pathogenesis Activity, Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention

2Tick-Borne Diseases Activity, Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention

Immunology, Issue 60, Ixodes scapularis, Lyme disease, Borrelia burgdorferi, salivary glands, hemolymph, tick dissection, saliva, tick,
Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Launch Video Published: 02/21/2012 ID: 3466
Oksana Polesskaya1
Anita Sun2
Gheorghe Salahura2
Jharon N. Silva1
Stephen Dewhurst1
Karl Kasischke3

1Department of Microbiology and Immunology, University of Rochester Medical Center

2Center for Neural Development and Disease, University of Rochester Medical Center

3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center

Neuroscience, Issue 60, mouse, two-photon, cortex, nicotinamide adenine dinucleotide, angiography, hypoxia,
Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Launch Video Published: 02/20/2012 ID: 3416
Urszula Polak1, 2
Calley Hirsch1
Sherman Ku3
Joel Gottesfeld3
Sharon Y.R. Dent1
Marek Napierala1

1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center

2Department of Cell Biology, Poznan University of Medical Sciences

3Department of Molecular Biology, The Scripps Research Institute

Developmental Biology, Issue 60, stem cells, induced pluripotent stem cells, iPSC, somatic cell reprogramming, pluripotency, retroviral transduction,
A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Launch Video Published: 02/20/2012 ID: 3265
Leland J. Cseke1
Sharon M. Talley2

1Department of Biological Sciences, The University of Alabama, Huntsville

2USDA-APHIS-PPQ, Center for Plant Health Science and Technology

Molecular Biology, Issue 60, Molecular genotyping, Japanese blood grass, Red Baron, cogongrass, invasive plants,
Morphometric Analyses of Retinal Sections

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Launch Video Published: 02/19/2012 ID: 3377
Tin Fung Chan1
Kin Chiu1
Carmen Ka Ming Lok1
Wing Lau Ho1
Kwok-Fai So1, 2, 3
Raymond Chuen-Chung Chang1, 2, 3

1Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong

2Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong

3State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong

Neuroscience, Issue 60, morphometric analysis, retina, thickness, cell size, Stereo Investigator, neuroscience,
Preterm EEG: A Multimodal Neurophysiological Protocol

This video explains the background theory of the neonatal EEG activity and the sensory responses, followed by a live demonstration of their recording in neonatal intensive care unit.

Launch Video Published: 02/18/2012 ID: 3774
Susanna Stjerna1
Juha Voipio2
Marjo Metsäranta3
Kai Kaila2, 4
Sampsa Vanhatalo1

1Department of Children's Clinical Neurophysiology, Helsinki University Hospital, University of Helsinki

2Department of Biosciences, University of Helsinki

3Department of Pediatrics, Helsinki University Hospital, University of Helsinki

4Neuroscience Center, University of Helsinki

Neuroscience, Issue 60, neurophysiology, preterm baby, neonatal, EEG, evoked response, high density EEG, FbEEG, sensory evoked response, neonatal intensive care unit,
In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Launch Video Published: 02/17/2012 ID: 3888
Rachel A. Davidowitz
Marcin P. Iwanicki
Joan S. Brugge

Department of Cell Biology, Harvard Medical School

Medicine, Issue 60, Ovarian Cancer, Metastasis, In vitro Model, Mesothelial, Spheroid,
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Launch Video Published: 02/17/2012 ID: 3661
Mansoureh Sameni1
Arulselvi Anbalagan1
Mary B. Olive1
Kamiar Moin1, 2
Raymond R. Mattingly1, 2
Bonnie F. Sloane1, 2

1Department of Pharmacology, Wayne State University

2Barbara Ann Karmanos Cancer Institute, Wayne State University

Medicine, Issue 60, Immunology, Breast, cancer, extracellular matrix, invasion, proteolysis, tumor microenvironment,