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Developmental Biology

Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis

Featured protocol,  Authors: Imre Gáspár
Imre GáspárAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: imre.gaspar@embl.de
Bio-protocol author page: a4784
 and Anne Ephrussi
Anne EphrussiAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: anne.ephrussi@embl.de
Bio-protocol author page: a4785
date: 7/5/2017, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2380.

Brief version appeared in EMBO J, Feb 2017
Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

Oxidative Stress Assays (arsenite and tBHP) in Caenorhabditis elegans

Featured protocol,  Authors: Collin Yvès Ewald
Collin Yvès EwaldAffiliation 1: Department of Health Sciences and Technology, Eidgenössische Technische Hochschule (ETH) Zürich, Schwerzenbach-Zürich, Switzerland
Affiliation 2: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 3: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 4: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
For correspondence: collin-ewald@ethz.ch
Bio-protocol author page: a4749
John M. Hourihan
John M. HourihanAffiliation 1: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 2: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 3: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
Bio-protocol author page: a4750
 and T. Keith Blackwell
T. Keith BlackwellAffiliation 1: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 2: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 3: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
Bio-protocol author page: a4751
date: 7/5/2017, 254 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2365.

Brief version appeared in Elife, Jan 2017
Cells and organisms face constant exposure to reactive oxygen species (ROS), either from the environment or as a by-product from internal metabolic processes. To prevent cellular damage from ROS, cells have evolved detoxification mechanisms. The activation of these detoxification mechanisms and their downstream responses represent an overlapping defense response that can be tailored to different sources of ROS to adequately adapt and protect cells. In this protocol, we describe how to measure the sensitivity to oxidative stress from two different sources, arsenite and tBHP, using the nematode C. elegans.

Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis

Authors: Imre Gáspár
Imre GáspárAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: imre.gaspar@embl.de
Bio-protocol author page: a4784
 and Anne Ephrussi
Anne EphrussiAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: anne.ephrussi@embl.de
Bio-protocol author page: a4785
date: 7/5/2017, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2380.

[Abstract] Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate ...

Oxidative Stress Assays (arsenite and tBHP) in Caenorhabditis elegans

Authors: Collin Yvès Ewald
Collin Yvès EwaldAffiliation 1: Department of Health Sciences and Technology, Eidgenössische Technische Hochschule (ETH) Zürich, Schwerzenbach-Zürich, Switzerland
Affiliation 2: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 3: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 4: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
For correspondence: collin-ewald@ethz.ch
Bio-protocol author page: a4749
John M. Hourihan
John M. HourihanAffiliation 1: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 2: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 3: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
Bio-protocol author page: a4750
 and T. Keith Blackwell
T. Keith BlackwellAffiliation 1: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 2: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 3: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
Bio-protocol author page: a4751
date: 7/5/2017, 254 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2365.

[Abstract] Cells and organisms face constant exposure to reactive oxygen species (ROS), either from the environment or as a by-product from internal metabolic processes. To prevent cellular damage from ROS, cells have evolved detoxification mechanisms. The activation of these detoxification mechanisms and their downstream responses represent an overlapping defense ...

Validating Candidate Congenital Heart Disease Genes in Drosophila

Authors: Jun-yi Zhu*
Jun-yi ZhuAffiliation: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Bio-protocol author page: a4710
Yulong Fu*
Yulong FuAffiliation: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Bio-protocol author page: a4711
Adam Richman
Adam RichmanAffiliation: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Bio-protocol author page: a4712
 and Zhe Han
Zhe HanAffiliation 1: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Affiliation 2: Department of Pediatrics, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
For correspondence: zhan@childrensnational.org
Bio-protocol author page: a4713
 (*contributed equally to this work) date: 6/20/2017, 227 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2350.

[Abstract] Genomic sequencing efforts can implicate large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system to validate candidate gene association with pathology is therefore useful. We present such a system employing Drosophila to validate candidate congenital heart disease (CHD) genes. The protocols ...

Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP)

Authors: Miguel Bernabé-Rubio
Miguel Bernabé-RubioAffiliation: Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain
Bio-protocol author page: a4399
David C. Gershlick
David C. GershlickAffiliation: Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, USA
Bio-protocol author page: a4400
 and Miguel A. Alonso
Miguel A. AlonsoAffiliation: Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain
For correspondence: maalonso@cbm.csic.es
Bio-protocol author page: a4401
date: 4/20/2017, 551 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2244.

[Abstract] In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove the ...

In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis

Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 4/5/2017, 573 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2215.

[Abstract] Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal ...

In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans

Authors: Jun Takayama
Jun TakayamaAffiliation: RIKEN Quantitative Biology Center, Laboratory for Developmental Dynamics, Kobe, Japan
Bio-protocol author page: a4310
Masashi Fujita
Masashi FujitaAffiliation: RIKEN Quantitative Biology Center, Laboratory for Developmental Dynamics, Kobe, Japan
Bio-protocol author page: a4311
 and Shuichi Onami
Shuichi OnamiAffiliation: RIKEN Quantitative Biology Center, Laboratory for Developmental Dynamics, Kobe, Japan
For correspondence: sonami@riken.jp
Bio-protocol author page: a4312
date: 4/5/2017, 766 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2205.

[Abstract] Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of ...

Assessment of Murine Retinal Function by Electroretinography

Authors: Gillie Benchorin
Gillie BenchorinAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
Bio-protocol author page: a4327
Melissa A. Calton
Melissa A. CaltonAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
Bio-protocol author page: a4328
Marielle O. Beaulieu
Marielle O. BeaulieuAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
Bio-protocol author page: a4329
 and Douglas Vollrath
Douglas VollrathAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
For correspondence: vollrath@stanford.edu
Bio-protocol author page: a4330
date: 4/5/2017, 601 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2218.

[Abstract] The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal ...

Measuring Caenorhabditis elegans Sleep during the Transition to Adulthood Using a Microfluidics-based System

Authors: Huiyan Huang
Huiyan HuangAffiliation: Department of Neuroscience, Brown University, Providence, USA
Bio-protocol author page: a4251
Komudi Singh
Komudi SinghAffiliation: Department of Neuroscience, Brown University, Providence, USA
Present address: Laboratory of Mitochondrial Biology & Metabolism, National Institute of Health, Bethesda, USA
Bio-protocol author page: a4252
 and Anne C. Hart
Anne C. HartAffiliation: Department of Neuroscience, Brown University, Providence, USA
For correspondence: anne_hart@brown.edu
Bio-protocol author page: a4253
date: 3/20/2017, 705 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2174.

[Abstract] C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski and Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During ...

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Authors: Nikos Kourtis
Nikos KourtisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Pathology, NYU School of Medicine, New York, USA
Bio-protocol author page: a4141
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 3/5/2017, 884 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2156.

[Abstract] Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals....

Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin

Authors: Eri Shibata
Eri ShibataAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Bio-protocol author page: a4049
Kazunori Ando
Kazunori AndoAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Bio-protocol author page: a4050
 and Atsushi Kawakami
Atsushi KawakamiAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: atkawaka@bio.titech.ac.jp
Bio-protocol author page: a4051
date: 1/20/2017, 965 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2109.

[Abstract] Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful approach for tracking and ...
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[Bio101] In vivo BrdU Incorporation and Detection in Mouse

Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, date: 6/5/2011, 20809 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.81.

[Abstract] BrdU (Bromodeoxyuridine or 5-bromo-2’-deoxyuridine) is a synthetic nucleoside that is incorporated into DNA by proliferating cells. This protocol is to be used to incorporate and detect BrdU in murine plasma cells. The plasma cells described in this protocol are formed spontaneously in autoimmune mice ...

Alcian Blue – Alizarin Red Staining of Mouse Skeleton

Author: Peichuan Zhang
Peichuan ZhangAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
date: 4/20/2012, 16661 views, 9 Q&A
DOI: https://doi.org/10.21769/BioProtoc.162.

[Abstract] Our lab has used the Alcian blue – Alizarin red staining method with certain modifications to characterize skeleton deformities in mice lacking Pek/Perk, encoding a translational control eIF2alpha kinase....

[Bio101] Lifespan Assay

Author: Fanglian He date: 4/5/2011, 15132 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.57.

[Abstract] This assay is used to address aging-related questions in worms....

[Bio101] Making Males of C. elegans

Author: Fanglian He date: 4/20/2011, 14229 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.58.

[Abstract] Getting males from a hermaphrodite population. This is a modified version of protocol originally written by Michael Koelle at Yale University....

Mouse Cochlear Whole Mount Immunofluorescence

Authors: Omar Akil
Omar AkilAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
For correspondence: oakil@ohns.ucsf.edu
Bio-protocol author page: a238
 and Lawrence R. Lustig
Lawrence R. LustigAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
Bio-protocol author page: a239
date: 3/5/2013, 11953 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.332.

[Abstract] This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest ...

c-Fos and Arc Immunohistochemistry on Rat Cerebellum

Author: Soyun Kim
Soyun KimAffiliation: Neuroscience Program, University of Southern California, Los Angeles, USA
For correspondence: soyunkimucsd@gmail.com
Bio-protocol author page: a45
date: 5/20/2012, 11412 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.191.

[Abstract] This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural ...

Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos

Authors: Michelle F. Goody
Michelle F. Goody Affiliation: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, USA
Bio-protocol author page: a619
 and Clarissa A. Henry
Clarissa A. HenryAffiliation: School of Biology and Ecology, University of Maine, Orono, USA
For correspondence: clarissa.henry@umit.maine.edu
Bio-protocol author page: a620
date: 6/5/2013, 11088 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.786.

[Abstract] Fluorescent conjugated Phalloidin is a stain that allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest. Here is a protocol for Phalloidin ...

[Bio101] Cell Culture Transfection for Production and Purification of Wnt Ligands

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 9907 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.176.

[Abstract] Wnt ligand proteins are extremely difficult to purify and enrich in vitro. This protocol uses Wnt11r protein as an example to illustrate how to use 293T cells to produce secreted Wnt11r and collect it in vitro for further biochemical experiments....

[Bio101] Mouse Embryonic Stem Cell Maintenance for Differentiation

Author: Hogune Im
Hogune ImAffiliation: Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical Schoo, Madison, USA
For correspondence: hoguneim@stanford.edu
Bio-protocol author page: a18
date: 10/20/2011, 9129 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.145.

[Abstract] Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a protocol to maintain mouse stem cells ...

[Bio101] Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line

Author: Yuqiong Pan date: 10/5/2011, 8806 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.142.

[Abstract] Using ATCC ES C57BL/6 as an example, it is shown here how to culture mouse embryonic stem (ES) cell line. The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008]. The ES cells were shown to populate the germ line of two host blastocyst ...
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