JoVE General
Immunocytochemistry: Human Neural Stem Cells
Department of Pathology, University of California, Irvine (UCI)
Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.
JoVE Immunology and Infection
Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
1Immunology, University of Toronto, 2Arthritis and Immune Disorder Research Centre, University Health Network (UHN)
This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.
JoVE Clinical and Translational Medicine
Mouse Eye Enucleation for Remote High-throughput Phenotyping
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University
The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.
JoVE Immunology and Infection
A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
1Department of Biological Sciences, University of Alberta, 2División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, 3Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México
Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei
JoVE Neuroscience
Whole Animal Perfusion Fixation for Rodents
1Biomedical Engineering, University of Michigan, 2Department of Neurological Surgery, University of Washington School of Medicine
Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.
JoVE General
Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University
The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.
JoVE General
Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine
We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.
JoVE General
Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine
Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).
JoVE Neuroscience
Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development
Department of Neuroscience and Physiology, SUNY Upstate Medical University
This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.
JoVE Immunology and Infection
Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging
Department of Cellular and Molecular Medicine, University of Bristol, UK
Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).
JoVE General
Flash Freezing and Cryosectioning E12.5 Mouse Brain
Department of Developmental and Cell Biology, University of California, Irvine (UCI)
Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.
JoVE General
Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.
JoVE General
Brain Banking: Making the Most of your Research Specimens
1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal
Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.
JoVE Clinical and Translational Medicine
Teratoma Generation in the Testis Capsule
1Department of Chemical Physiology, Scripps Research Institute, 2Department of Chemical Physiology, Scripps Research Institute, 3Department of Reproductive Medicine, University of California
Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.
JoVE Clinical and Translational Medicine
A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage
1Department of Neurosurgery, SUNY Upstate Medical University, 2Department of Neuroscience and Physiology, SUNY Upstate Medical University
Aneurysmal subarachnoid hemorrhage (SAH) is bleeding that occurs into the subarachnoid space when an aneurysm ruptures. While the morbidity and mortality from this event has been on a decline due to improved treatment approaches, the risk of vasospasm after subarachnoid hemorrhage continues to be the same as it was several years ago. The importance of establishing a comprehensive and reproducible animal model to identify initiating events of cerebral vasospasm has been the focus of research since the first use of rats in an experimental vasospasm model in 1979 by Barry et al. Early work in rats demonstrated that a single injection of autologous blood into the cisterna magna led to acute (within minutes) but not delayed cerebral vasospasm 3, 6, 14. Here we characterize a low mortality SAH rat model that results in reproducible delayed vasospasm.
JoVE Neuroscience
Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP
1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, Louisiana Tech University
Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.
JoVE Neuroscience
An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells
Department of Neuroscience, University of Minnesota
This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.
JoVE Bioengineering
Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound
1School of Biochemistry, University of Bristol, 2Smith and Nephew
This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.
JoVE Clinical and Translational Medicine
3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System
1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, 2Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, 3Department of Biology, Duke University
A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.
JoVE General
Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Department of Biochemistry, University of Toronto
Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.
JoVE Neuroscience
Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice
Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, University of Toledo
The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.
JoVE General
Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)
1Genome Technology Branch, National Human Genome Research Institute, 2Neuroscience and Cognitive Science Program, University of Maryland
The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.
JoVE General
Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University
This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.
JoVE General
Methods for the Study of the Zebrafish Maxillary Barbel
1Department of Biological Sciences, DePaul University, 2Children’s Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine
The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.
JoVE General
Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.
JoVE General
The Gateway to the Brain: Dissecting the Primate Eye
1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal, 3Departement de chimie-biologie, Universite du Quebec a Trois-Rivieres
The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.
JoVE General
Dissecting the Non-human Primate Brain in Stereotaxic Space
1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal, 3Département de chimie-biologie , Université du Québes à Trois-Rivières
The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.
JoVE Neuroscience
Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells
1Department of Molecular Genetics and Molecular Biology , Duke University, 2Department of Chemistry, Duke University
Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.
JoVE Neuroscience
Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue
1Department of Neurology, Beth Israel Deaconess Medical Center, 2Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 3Department of Pathology, Beth Israel Deaconess Medical Center, 4Department of Pathology, Division of Neuropathology, Brigham and Women's Hospital
A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.
JoVE Immunology and Infection
Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System
An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.
JoVE Neuroscience
Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis
1Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Department of Neurological Surgery, University of California, San Francisco, 3Department of Neurology, University of California, San Francisco
In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.
JoVE Clinical and Translational Medicine
The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse
1Department of Pharmacology and Physiology, University of Rochester, 2Department of Neurology, University of Alabama at Birmingham, 3Departments of Anesthesiology, Pharmacology and Physiology, University of Rochester
Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.
JoVE Clinical and Translational Medicine
Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)
1Dept. of Neurology, University Medicine Göttingen, 2DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany
A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.
JoVE Neuroscience
Chicken Embryo Spinal Cord Slice Culture Protocol
Research Department of Cell and Developmental Biology, University College London
Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.
JoVE General
In situ Protocol for Butterfly Pupal Wings Using Riboprobes
1Department of Biological Sciences, SUNY-University at Buffalo, 2Dept. Ecology and Evolutionary Biology, Yale University
In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.
JoVE General
Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies
Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.
JoVE Immunology and Infection
Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients
The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.
JoVE General
Dissection of the Adult Zebrafish Kidney
Department of Biological Sciences, University of Notre Dame
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
JoVE General
Drosophila Pupal Abdomen Immunohistochemistry
Department of Biological Sciences, University of Alabama
Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.
JoVE General
Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University, 2Department of Biological Sciences, Hokkaido University
This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.
JoVE Neuroscience
Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones
Department of Neuroscience, University of Minnesota
A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.
JoVE General
Single Cell Fate Mapping in Zebrafish
1Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 2Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center
A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.
JoVE General
FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability
1Biotechnology Research Institute, AUT University and KODE Biotech Ltd, 2Shemyakin Institute of Bioorganic Chemistry RAS, Moscow, Russia
Function-Spacer-Lipid (FSL) constructs allow the surface characteristics of living cells and virions to be modified without loss of vitality. The method requires only simple contact of an FSL construct solution with a cell/virion and spontaneous and stable surface incorporation occurs.
JoVE Neuroscience
Inducing Dendritic Growth in Cultured Sympathetic Neurons
Department of Molecular Biosciences, University of California, Davis
We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.
JoVE Neuroscience
Isolation and Culture of Hippocampal Neurons from Prenatal Mice
Department of Biological Sciences, Auburn University
We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.
JoVE Immunology and Infection
Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry
Institute for Molecular Infection Biology (IMIB), University of Würzburg
Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.
JoVE Application Notes
Abcam Quantitative Cleaved PARP-1 High-Throughput In-Cell ELISA (ICE) Assay - ADVERTISEMENT
Abcam, plc
Quantitative measurement of cleaved PARP-1 in fixed adherent or suspension cells by high-throughput In-Cell ELISA for using infra-red Li-Cor imaging system.
JoVE General
Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Department of Molecular Microbiology, Faculty of Medicine, Technion - Israel Institute of Technology
Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.
JoVE General
Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos
1Institut de Recherches Cliniques de Montréal (IRCM), 2Department of Biochemistry, Université de Montréal
Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.
JoVE General
RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs
Department of Biological Sciences, Union College
By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.
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