1Department of Basic Sciences, Mississippi State University College of Veterinary Medicine
The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.
Published February 6, 2015. Keywords: Neuroscience, Laser capture microdissection, dopamine neuron, Immunohistochemistry, Tyrosine hydroxylase, Ventral tegmental area, PEN membrane glass slide.
1Department of Neurosurgery, Brigham & Women's Hospital, Harvard Medical School, 2Division of Brain Sciences, Department of Medicine, Imperial College London
The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.
Published March 8, 2015. Keywords: Neuroscience, anterior thalamic nucleus, deep brain stimulation, dentate gyrus, hippocampus, epilepsy, gene expression, high-frequency stimulation, quantitative RT-PCR
1Department of Cell Biology, University of Massachusetts Medical School
We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.
Published April 29, 2011. Keywords: Developmental Biology, Myogenesis, Chromatin, Gene Regulation, Chromatin Immunoprecipitation, Embryo, Mouse
1Department of Hematology and Oncology, University of Illinois at Chicago, 2Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center
The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.
Published October 27, 2014. Keywords: Cellular Biology, Wnt, Frizzled, Soft Agar Assay, Colony Formation Assay, tumor suppressor, lung cancer
1Physical Biosciences Division, Lawrence Berkeley National Laboratory
This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.
Published July 21, 2014. Keywords: Genetics, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip
JoVE Immunology and Infection
1Department of Pathology and Molecule Medicine, McMaster University
Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.
Published January 17, 2014. Keywords: Immunology, Streptococcus pneumoniae, Nasal lavage, nasopharynx, murine, flow cytometry, RNA, Quantitative PCR, recruited macrophages, neutrophils, T-cells, effector cells, intranasal colonization
1Department of Biology, Johns Hopkins University
Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.
Published March 23, 2012. Keywords: Genetics, ChIP, Drosophila, testes, q-PCR, high throughput sequencing, epi-genetics
JoVE Immunology and Infection
1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine, 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin, 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin
This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens.
Published February 4, 2012. Keywords: Immunology, Influenza virus, Virus titer, Epithelial cells