Protocols

Books for Xenopus Research and Protocols

• Xenopus Protocols: Post-Genomic Approaches. Hoppler and Vize, 2012 [1]
  • expanded second edition with novel approaches inspired by X. tropicalis genome sequencing.

• The Laboratory Xenopus sp. Green, 2010 [2]
  • 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 [3]
  • step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents.

• Color Atlas of Xenopus laevis Histology, Wiechmann and Wirsig-Wiechmann, 2003 [4]
  • central source on the microscopic anatomy of cells, tissues, and major organs of Xenopus laevis.

• Early Development of Xenopus laevis: A Laboratory Manual, Sive, Grainger, and Harland, 2000 [5]
  • comprehensive collection of protocols for the study of early development in Xenopus embryos

• Transgenic Xenopus: Microinjection Methods and Developmental Neurobiology, Seidman and Soreq, 1997 [6]
  • 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 [7]
  • scanning, transmission, and light microscopy images of Xenopus embryonic development.

• Normal Table of Xenopus laevis (Daudin), Nieuwkoop and Faber (Third edition), 1994 [8]
  • a systematic and chronological description of Xenopus laevis development.

• Xenopus laevis: Practical Uses in Cell and Molecular Biology, Volume 36, Kay and Peng, 1991 [9]
  • detailed description of a wide range of uses for Xenopus laevis oocytes and embryos in cell and molecular biology.

• The early development of Xenopus laevis. An atlas of the histology, Hausen and Riebesell, 1991 [10]
  • detailed histological sections of Xenopus embryonic development

Online Xenopus Resources

External links to the Journal of Visualized Experiments (JOVE) - Xenopus embryo [11] - excellent video demonstrations

• • Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles - Terni et al. -[12]
  • Obtaining Eggs from Xenopus laevis Females - Cross & Powers [13]
  • Microinjection of Xenopus Laevis Oocytes - Cohen et al. - [14]
  • Fertilization of Xenopus oocytes, Host Transfer Method - Schneider et al. [15]
  • Tissue Determination Using Animal Cap Transplant (ACT) Assay in X. laevis - Viczian & Zuber [16]
  • Blastomere Explants to Test for Cell Fate Commitment - Grant et al. [17]
  • Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation - Amaya & Kroll [18]
  • Organizer and Animal Pole Explants from X. laevis Embryos and Cell Adhesion Assay - Ogata & Cho [19]
  • Plastic Embedding and Sectioning of X. laevis Embryos - Ogata1 et al. [20]
  • Visualizing RNA Localization in Xenopus Oocytes - Gagnon & Mowry [21]
  • Neural Explant Cultures from Xenopus laevis - Lowery et al. [22]
  • Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling - Itoh & Sokol [23]
  • Dissection, Culture, and Analysis of X. laevis Embryonic Retinal Tissue - McDonough et al. [24]
  • X. tropicalis Egg Extracts to Identify Microtubule-associated RNAs - Sharp & Blower [25]
  • Electroporation of Craniofacial Mesenchyme - Tabler & Liu [26]
  • Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures - Yazejian et al. [27]
  • Single Cell Electroporation in vivo within the Intact Developing Brain - Hewapathirane & Haas - [28]
  • Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells - Joshi & Davidson [29]
  • Preparation and Fractionation of Xenopus laevis Egg Extracts - Cross & Powers [30]
  • In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts - Cross & Powers [31]
  • Study of the DNA Damage Checkpoint using Xenopus Egg Extracts - Willis et al. [32]
  • Two Types of Assays for Detecting Frog Sperm Chemoattraction - Burnett et al. [33]
  • Comparative in vivo Study of gp96 Adjuvanticity in X. laevis - Nedelkovska et al. [34]
  • Patch Clamp and Perfusion Techniques - Yang et al. [35]
  • Patch Clamp Recording of Ion Channels - Brown et al. [36]
  • Cation Transport in Xenopus Oocytes - Dürr et al.[37]
  • Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo - BC.Durand [38]
  • Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation - Millet & Monsoro-Burq [39]

Cold Spring Harbor Xenopus Protocols [40] - external links provided as subscription is required for access to some protocols. 2019 and 2020 articles listed above, earlier protocols below.

All articles are Copyright 2019  Cold Spring Harbor Laboratory Press, or Copyright 2020  Cold Spring Harbor Laboratory Press- please cite and request permission to re-use images/figures accordingly.

• • Raising Antibodies for Use in Xenopus. Maya Z. Piccinni and Matthew J. Guille. 2020. [41]
  • Purifying Antibodies Raised against Xenopus Peptides. Maya Z. Piccinni and Matthew J. Guille. 2020. [42]
  • Assessing the Immune Response When Raising Antibodies for Use in Xenopus. Maya Z. Piccinni and Matthew J. Guille. 2020. [43].
  • In Vitro Transcription Systems. Michael R. Green and Joseph Sambrook. [44]
  • Applying Tensile and Compressive Force to Xenopus Animal Cap Tissue. Georgina K. Goddard, Nawseen Tarannum, and Sarah Woolner. 2020. [45].
  • Transcriptomics and Proteomics Methods for Xenopus Embryos and Tissues. Michael J. Gilchrist, Gert Jan C. Veenstra, and Ken W.Y. Cho. 2020. ''Topic Introduction'' [46]
  • In Vivo Assessment of Neural Precursor Cell Cycle Kinetics in the Amphibian Retina. Morgane Locker and Muriel Perron2019. [47], Supplemental Material [48]
  • Following Endocrine-Disrupting Effects on Gene Expression in Xenopus laevis. Petra Spirhanzlova, Michelle Leemans, Barbara A. Demeneix, and Jean-Baptiste Fini. 2019. Full-text link: [49].
  • Analysis of Chromatin Binding of Ectopically Expressed Proteins in Early Xenopus Embryos. Laura J.A. Hardwick and Anna Philpott. 2019. [50].
  • Analysis of Phosphorylation Status of Ectopically Expressed Proteins in Early Xenopus Embryos. Laura J.A. Hardwick and Anna Philpott. 2019. [51].
  • An RNA-Seq Protocol for Differential Expression Analysis. Nick D.L. Owens, Elena De Domenico, and Michael J. Gilchrist. 2019. [52]
  • Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems. Gary S. McDowell and Anna Philpott. 2019. [53].
  • The Use of Cell-Free Xenopus Extracts to Investigate Cytoplasmic Events. Romain Gibeaux and Rebecca Heald. 2019.''Topic Introduction'' [54]
  • Mass Spectrometry-Based Absolute Quantification of Single Xenopus Embryo Proteomes. Rik G.H. Lindeboom, Arne H. Smits, Matteo Perino, Gert Jan C. Veenstra, and Michiel Vermeulen. [55]
  • Assessing Ubiquitylation of Individual Proteins Using Xenopus Extract Systems. Gary S. McDowell and Anna Philpott. 2019. [56]
  • INTACT Proteomics in Xenopus. Lauren Wasson, Nirav M. Amin, and Frank L. Conlon. 2019. [57]
  • Special Considerations for Making Explants and Transplants with Xenopus tropicalis. 2019. Marilyn Fisher and Robert M. Grainger. [58]. Supplemental Material [59].
  • Skin Grafting in Xenopus laevis: A Technique for Assessing Development and Immunological Disparity. Yumi Izutsu. 2019. [60]
  • Adoptive Transfer of Fluorescently Labeled Immune Cells in Xenopus. Kun Hyoe Rhoo and Jacques Robert. [61]
  • Generating a Three-Dimensional Genome from Xenopus with Hi-C. Ian K. Quigley and Sven Heinz. 2019. [62]
  • Assessing Antibody Responses to Pathogens or Model Antigens in Xenopus by Enzyme-Linked Immunosorbent Assay (ELISA). Francisco De Jesús Andino and Jacques Robert. 2019 [63]
  • DNase-seq to Study Chromatin Accessibility in Early Xenopus tropicalis Embryos. Jin Sun Cho, Ira L. Blitz, and Ken W.Y. Cho. 2019 [64]
  • Studies of Limb Regeneration in Larval Xenopus. Anthony L. Mescher and Anton W. Neff. 2019. [65].
  • Ex Vivo Eye Tissue Culture Methods for Xenopus. Jonathan J. Henry, Kimberly J. Perry, and Paul W. Hamilton. 2019. [66].
  • Methods for Examining Lens Regeneration in Xenopus. Jonathan J. Henry, Kimberly J. Perry, and Paul W. Hamilton. 2019. [67]
  • Mapping Chromatin Features of Xenopus Embryos. George E. Gentsch and James C. Smith. 2019. [68]
  • Reconstituting Nuclear and Chromosome Dynamics Using Xenopus Extracts.

.

2019/2020 CSHL Recipes

• • Embryo Lysis Buffer (Xenopus). (Recipe 1) CSHLP. 2019 [69]
  • Xenopus Embryo Lysis Buffer. (Recipe 2). CSHLP. 2019 [70]
  • DNA Isolation Buffer. CSHLP. 2019 [71]
  • Lysis Buffer for Xenopus Hi-C. CSHLP 2019. [72]
  • RIPA Buffer for Xenopus. CSHLP 2019. [73]
  • Amphibian Serum-Free (ASF) Medium Supplemented with Fetal Bovine Serum (FBS). CSHL 2019. [74]
  • Xenopus Eye Culture Medium CSHLP 2019. [75]
  • Marc's Modified Ringer's (MMR) for Xenopus (20×). CSHLP. 2019. [76]

Cold Spring Harbor Xenopus Protocols 2007. Edited by Hazel Sive

• • Housing and Feeding of Xenopus laevis - Sive et al. [77]
  • Inducing Ovulation in Xenopus laevis - Sive et al. [78]
  • Xenopus laevis In Vitro Fertilization and Natural Mating Methods - Sive et al. [79]
  • Egg Collection and In Vitro Fertilization of the Western Clawed Frog Xenopus tropicalis - Showell & Conlon [80]
  • Isolation of Xenopus Oocytes - Sive et al. [81]
  • Isolating Xenopus laevis Testes - Sive et al. [82]
  • Dejellying Xenopus laevis Embryos - Sive et al. [83]
  • Removing the Vitelline Membrane from Xenopus laevis Embryos - Sive et al. [84]
  • Microinjection of Xenopus Embryos - Sive et al. [85]
  • Defolliculation of Xenopus Oocytes - Sive et al. [86]
  • Microinjection of Xenopus Oocytes - Sive et al. [87]
  • Animal Cap Isolation from Xenopus laevis - Sive et al. [88]
  • Xenopus laevis Keller Explants - Sive et al. [89]
  • Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes - Sive et al. [90]
  • Calibration of the Injection Volume for Microinjection of Xenopus Oocytes and Embryos - Sive et al. [91]
  • Isolation of DNA from Red Blood Cells in Xenopus - Sive et al. [92]
  • Investigating Morphogenesis in Xenopus Embryos: Imaging Strategies, Processing, and Analysis - Kim & Davidson [93]
  • Low-Magnification Live Imaging of Xenopus Embryos for Cell and Developmental Biology - Wallingford [94]
  • High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology - Keiserman et al. [95]
  • Preparation of Fixed Xenopus Embryos for Confocal Imaging - Wallingford [96]
  • Whole-Mount Fluorescence Immunocytochemistry on Xenopus Embryos - Lee et al. [97]
  • Generation of Transgenic Xenopus laevis - Kroll & Amaya - [98] [99] [100]
  • In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus - Ruthazer et al. [101]
  • Photoconversion for Tracking the Dynamics of Cell Movement in Xenopus laevis Embryos - Chernet et al. [102]
  • Single-Cell Electroporation in Xenopus - Liu & Haas [103]
  • Imaging Axon Pathfinding in Xenopus In Vivo - Leung & Holt [104]
  • A Versatile Protocol for mRNA Electroporation of Xenopus laevis Embryos - Chernet & Levin [105]

General Research Protocols

Animal Husbandry

• obtain frogs and frog housing equipment [106]
• Xenopus Express links [107] - Xenopus Express husbandry [108]
• Xenopus tropicalis and Xenopus laevis environmental parameter standard operating procedure (Kroll lab)

Lab Solutions and Reagents (click each to view expanded content)

• 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)

Transgenesis

• 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). [109]
• A method for generating transgenic frog embryos. Ishibashi S, Kroll KL, Amaya E. Methods Mol Biol. 2008;461:447-66. [110]
• A method for generating transgenic frog embryos. Amaya E, Kroll KL. Methods Mol Biol. 1999;97:393-414. [111]

in situ Hybridization

• in situ hybridization, brief version (Harland lab)
• whole-mount in situ hybridization (Conlon lab)
  • in situ checklist (Conlon lab)
• whole-mount in situ hybridization - LNA probes (Wheeler lab)
• fluorescent in-situs and FCIS (Vize lab)
  • Wholemount embryo FISH / Double FISH
  • Flourescin Tyramide Synthesis
  • Cy3 Tyramide Synthesis
  • FCIS
• RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA) (Myers and Scarlett labs)

Immunohistochemistry

• 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)
• RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA) (Myers and Scarlett labs)

ChIP protocols

• Chromatin immunoprecipitation analysis of Xenopus embryos., Methods Mol Biol. 2012;917:279-92. [112] [113]
  • Akkers RC, Jacobi UG, Veenstra GJ.
• Chromatin immunoprecipitation in early Xenopus laevis embryos., Dev Dyn. 2009 Jun;238(6):1422-32. [114] [115]
  • Blythe SA, Reid CD, Kessler DS, Klein PS.

Histology

• cryosectioning Xenopus embryos (Conlon lab)

Embryo Staining Protocols (non in situ)

• 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)

Immuno 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)
• frozen egg extracts that retain spindle assembly activity (Takagi and Shimamoto, 2017)

Morpholino Studies in Xenopus Brain Development

Bestman and Cline 2019, Brain Development pp 377-395, Part of the Methods in Molecular Biology book series (MIMB, volume 2047) [[116]]

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)