NIH 6 articles published in JoVE Developmental Biology Optogenetic Signaling Activation in Zebrafish Embryos Allison J. Saul*1, Catherine E. Rogers*1, Marcial Garmendia-Cedillos2, Thomas Pohida2, Katherine W. Rogers1 1Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, 2Instrumentation Development and Engineering Application Solutions, National Institute of Biomedical Imaging and Bioengineering, NIH Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo. Cancer Research Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia Gisele O. L. Rodrigues1, WenQing Li1, Sarah D. Cramer1,2,3, Hila Y. Winer1, Tu Chun Hsu1,2,3, Timothy Gower4, Julie A. Hixon1, Scott K. Durum1 1Cytokines and Immunity Section, Cancer Innovation Laboratory, National Cancer Institute, National Institutes of Health, 2Comparative Biomedical Scientist Training Program, NIH, 3Department of Pathobiology and Diagnostic Investigation, Veterinary Diagnostic Laboratory, Michigan State University, 4NCI-Frederick Laboratory Animal Sciences Program This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis. Immunology and Infection An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions Jansy P. Sarathy1, Hsin-pin Ho Liang1, Danielle Weiner2, Jacqueline Gonzales2, Laura E. Via2, Véronique Dartois1 1Public Health Research Institute Centre, New Jersey Medical School, Rutgers, 2Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum. Neuroscience Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue F. Javier Rubio*1, Xuan Li*1, Qing-Rong Liu1, Raffaello Cimbro2, Bruce T. Hope1 1Behavioral Neuroscience Research Branch, Intramural Research Program, NIDA, NIH, DHHS, 2Division of Rheumatology, School of Medicine, Johns Hopkins University Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects. Immunology and Infection Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface Marcia Arenas-Hernandez1, Elly N. Sanchez-Rodriguez1, Tara N. Mial1, Sarah A. Robertson2, Nardhy Gomez-Lopez1,3,4 1Department of Obstetrics & Gynecology, Wayne State University School of Medicine, 2School of Paediatrics and Reproductive Health, Research Centre for Reproductive Health, the Robinson Research Institute, The University of Adelaide, 3Department of Immunology & Microbiology, Wayne State University School of Medicine, 4Perinatology Research Branch, NICHD/NIH/DHHS Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis. Immunology and Infection Isolation of Leukocytes from the Human Maternal-fetal Interface Yi Xu1, Olesya Plazyo1, Roberto Romero1,2,3,4, Sonia S. Hassan1,5, Nardhy Gomez-Lopez1,5,6 1Perinatology Research Branch, NICHD/NIH/DHHS, 2Department of Obstetrics and Gynecology, University of Michigan, 3Department of Epidemiology and Biostatistics, Michigan State University, 4Department of Molecular Obstetrics and Genetics, Wayne State University, 5Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 6Department of Immunology and Microbiology, Wayne State University School of Medicine Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.
