Protocols

• Online Xenopus Resources
  • Cold Spring Harbor Protocols - Xenopus [1], subscription required for 130+ articles, select articles shown below:
    • Housing and Feeding of Xenopus laevis - Sive et al. [2]
    • Inducing Ovulation in Xenopus laevis - Sive et al. [3]
    • Xenopus laevis In Vitro Fertilization and Natural Mating Methods - Sive et al. [4]
    • Egg Collection and In Vitro Fertilization of the Western Clawed Frog Xenopus tropicalis - Showell & Conlon [5]
    • Isolation of Xenopus Oocytes - Sive et al. [6]
    • Isolating Xenopus laevis Testes - Sive et al. [7]
    • Dejellying Xenopus laevis Embryos - Sive et al. [8]
    • Removing the Vitelline Membrane from Xenopus laevis Embryos - Sive et al. [9]
    • Microinjection of Xenopus Embryos - Sive et al. [10]
    • Defolliculation of Xenopus Oocytes - Sive et al. [11]
    • Microinjection of Xenopus Oocytes - Sive et al. [12]
    • Animal Cap Isolation from Xenopus laevis - Sive et al. [13]
    • Xenopus laevis Keller Explants - Sive et al. [14]
    • Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes - Sive et al. [15]
    • Calibration of the Injection Volume for Microinjection of Xenopus Oocytes and Embryos - Sive et al. [16]
    • Isolation of DNA from Red Blood Cells in Xenopus - Sive et al. [17]
    • Investigating Morphogenesis in Xenopus Embryos: Imaging Strategies, Processing, and Analysis - Kim & Davidson [18]
    • Low-Magnification Live Imaging of Xenopus Embryos for Cell and Developmental Biology - Wallingford [19]
    • High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology - Keiserman et al. [20]
    • Preparation of Fixed Xenopus Embryos for Confocal Imaging - Wallingford [21]
    • Whole-Mount Fluorescence Immunocytochemistry on Xenopus Embryos - Lee et al. [22]
    • Generation of Transgenic Xenopus laevis - Kroll & Amaya - [23] [24] [25]
    • In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus - Ruthazer et al. [26]
    • Photoconversion for Tracking the Dynamics of Cell Movement in Xenopus laevis Embryos - Chernet et al. [27]
    • Single-Cell Electroporation in Xenopus - Liu & Haas [28]
    • Imaging Axon Pathfinding in Xenopus In Vivo - Leung & Holt [29]
    • A Versatile Protocol for mRNA Electroporation of Xenopus laevis Embryos - Chernet & Levin [30]

• • The Journal of Visualized Experiments (JOVE) - Xenopus embryo [31] - excellent video demonstrations
    • Obtaining Eggs from Xenopus laevis Females - Cross & Powers [32]
    • Microinjection of Xenopus Laevis Oocytes - Cohen et al. - [33]
    • Fertilization of Xenopus oocytes, Host Transfer Method - Schneider et al. [34]
    • Tissue Determination Using Animal Cap Transplant (ACT) Assay in X. laevis - Viczian & Zuber [35]
    • Blastomere Explants to Test for Cell Fate Commitment - Grant et al. [36]
    • Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation - Amaya & Kroll [37]
    • Organizer and Animal Pole Explants from X. laevis Embryos and Cell Adhesion Assay - Ogata & Cho [38]
    • Plastic Embedding and Sectioning of X. laevis Embryos - Ogata1 et al. [39]
    • Visualizing RNA Localization in Xenopus Oocytes - Gagnon & Mowry [40]
    • Neural Explant Cultures from Xenopus laevis - Lowery et al. [41]
    • Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling - Itoh & Sokol [42]
    • Dissection, Culture, and Analysis of X. laevis Embryonic Retinal Tissue - McDonough et al. [43]
    • X. tropicalis Egg Extracts to Identify Microtubule-associated RNAs - Sharp & Blower [44]
    • Electroporation of Craniofacial Mesenchyme - Tabler & Liu [45]
    • Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures - Yazejian et al. [46]
    • Single Cell Electroporation in vivo within the Intact Developing Brain - Hewapathirane & Haas - [47]
    • Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells - Joshi & Davidson [48]
    • Preparation and Fractionation of Xenopus laevis Egg Extracts - Cross & Powers [49]
    • In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts - Cross & Powers [50]
    • Study of the DNA Damage Checkpoint using Xenopus Egg Extracts - Willis et al. [51]
    • Two Types of Assays for Detecting Frog Sperm Chemoattraction - Burnett et al. [52]
    • Comparative in vivo Study of gp96 Adjuvanticity in X. laevis - Nedelkovska et al. [53]
    • Patch Clamp and Perfusion Techniques - Yang et al. [54]
    • Patch Clamp Recording of Ion Channels - Brown et al. [55]
    • Cation Transport in Xenopus Oocytes - Dürr et al.[56]

• Books for Xenopus Research and Protocols
  • Normal Table of Xenopus Laevis (Daudin), Nieuwkoop and Faber (Third edition), 1994 [57]
    • a systematic and chronological description of Xenopus laevis development
  • Early Development of Xenopus laevis: A Laboratory Manual, Sive, Grainger, and Harland, 2000 [58]
    • comprehensive collection of protocols for the study of early development in Xenopus embryos
  • The Laboratory Xenopus sp., Green, 2010 [59]
    • a highly detailed manual containing Xenopus husbandry, management, veterinary care, and frog and equipment vendor information
  • Xenopus Protocols: Cell Biology and Signal Transduction, Liu (First edition), 2006 [60]
    • step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents
  • Xenopus laevis: Practical Uses in Cell and Molecular Biology, Volume 36, Kay and Peng, 1991 [61]
    • detailed description of a wide range of uses for Xenopus laevis oocytes and embryos in cell and molecular biology
  • Transgenic Xenopus: Microinjection Methods and Developmental Neurobiology, Seidman and Soreq, 1997 [62]
    • referenced guide to the use of microinjected embryos studying the role and regulation of nervous system proteins during development
  • Atlas of Xenopus Development, Bernardini, Prati, Bonetti, and Scari, 1999 [63]
    • scanning, transmission, and light microscopy images of Xenopus embryonic development
  • The early development of Xenopus laevis. An atlas of the histology, Hausen and Riebesell, 1991 [64]
    • detailed histological sections of Xenopus embryonic development
  • Color Atlas of Xenopus laevis Histology, Wiechmann and Wirsig-Wiechmann, 2003 [65]
    • central source on the microscopic anatomy of cells, tissues, and major organs of Xenopus laevis

• Animal Husbandry
  • obtain frogs and frog housing equipment [66]
  • Xenopus Express links [67] - Xenopus Express husbandry [68]
  • Xenopus tropicalis and Xenopus laevis environmental parameter standard operating procedure (Kroll lab)

• Lab Solutions and Reagents - click to view expanded list
  • culture media
  • immunohistochemistry
  • in situ hybridization
  • fixatives
  • DNA/RNA preparation
  • embryo manipulation/dissection
  • microinjection
  • general buffers and salt solutions

• Generating Embryos
  • inducing egg laying via hCG injection (Vize lab)
  • anesthesia, in vitro fertilization, and natural mating of Xenopus tropicalis (Conlon lab)
  • benzocaine stock, 100x (Khokha lab)
  • in vitro fertilization of Xenopus laevis (Conlon lab)
  • high-salt solution for egg laying (Zorn lab/Ron Laskey)
  • dejellying embryos (Sive et al. 2000)
  • dejellying embryos (Zorn lab)

• Transgenics
  • transgenesis in Xenopus laevis - adapted from Amaya et al 2001 (Conlon lab)
  • tadpole tail sampling for genomic DNA (Conlon lab)
  • Production of transgenic Xenopus laevis by restriction enzyme mediated integration and nuclear transplantation. Amaya E, Kroll K. J Vis Exp. 2010 Aug 21;(42). [69]
  • A method for generating transgenic frog embryos. Ishibashi S, Kroll KL, Amaya E. Methods Mol Biol. 2008;461:447-66. [70]
  • A method for generating transgenic frog embryos. Amaya E, Kroll KL. Methods Mol Biol. 1999;97:393-414. [71]

• In Situ Hybridization
  • in situ hybridization, brief version (Harland lab)
  • whole-mount in situ hybridization (Conlon lab)
    • in situ checklist (Conlon lab)
  • fluorescent in-situs and FCIS (Vize lab)
    • Wholemount embryo FISH / Double FISH
    • Flourescin Tyramide Synthesis
    • Cy3 Tyramide Synthesis
    • FCIS

• Immunohisochemistry
  • wholemount antibody staining (CSH Protocols) (Wallingford lab)
  • wholemount antibody staining - alkaline phosphatase detection (Conlon lab)
  • wholemount antibody staining - adapted from Dan Weeks' protocol (Conlon lab)
  • immunostaining sectioned embryos (Conlon lab)
  • hydrochloric acid antigen retrieval (Zorn lab)

• Histology
  • cryosectioning Xenopus embryos (Conlon lab)

• Other Embryo Staining Protocols
  • benzidine-peroxidase staining for blood (Conlon lab)
  • benzidine-peroxidase staining for blood (Thomsen lab)
  • apoptosis via TUNEL (Veenstra lab)
  • TUNEL staining of whole Xenopus embryos - Harland protocol (Conlon lab)

• Other Immuno Protocols and Protein Protocols
  • immunoprecipitation (Stukenberg lab)
  • immunofluorescence with biotinylated antibodies (Stukenberg lab)
  • Purifying Polyclonal Antibodies using Affinity Columns/Chromatography
    • Making an Affinity Column
    • Running an Affinity Column
  • whole embryo protein extraction with freon (Conlon lab)
  • SDS-PAGE gels (Conlon lab)
  • western blot protocol (Conlon lab)
  • silver staining protein gels (Stukenberg lab)

• Nucleic Acid Protocols
  • RNA Isolation
    • Preparation of RNA Using Proteinase K/LiCl (Sive et al. 2000)
    • Preparation of RNA (In Vitro Transcription) (Sive et al. 2000)
  • DNA Isolation
    • Extraction of DNA from Single Embryos (Sive et al. 2000)
    • Isolation of DNA from Red Blood Cells (Sive et al. 2000)

• Oocyte Transfer Technique (Heasman/Wylie labs)
  • Part 1: Choosing Oligodeoxyribonucleotides (ODNs)
  • Part 2: ODN Modification
  • Part 3: Host Transfer Technique
  • Part 4: Fertilization and Development

• Xenopus Oocyte and Egg Extracts
  • crude interphase egg extract (Shechter lab)
  • isolation of chromatin from egg extract and histone recovery (Shechter lab)
  • preparation of cycling egg extract (Shechter lab)
  • cycling extracts, mike's protocol (Stukenberg lab)
  • cycling extracts, sara's protocol (Stukenberg lab)
  • CSF extract, mike's protocol (Stukenberg lab)
  • CSF extract, sara's protocol (Stukenberg lab)
  • assaying H1 kinase in extracts (Stukenberg lab)

• Xenopus Tissue Culture
  • tissue culture of Xenopus cell lines S3, XTC (Stukenberg lab)
  • isolation of mitotic chromosomes from XTC cells (Stukenberg lab)
  • in-vitro kinetochore-microtubule binding assays (Stukenberg lab)