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]

Protocols published in Journals

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) [[40]]

Cold Spring Harbor Xenopus Protocols 2018, 2019 and 2020. [41]

Note: External links are provided but a subscription fee is required for access most CSHL protocols.
All CSHL protocol articles are Copyright 2018, 2019, or 2020  Cold Spring Harbor Laboratory Press- Request permission to re-use images/figures accordingly.

General Research Protocols

• • Oocyte Host-Transfer and Maternal mRNA Depletion Experiments in Xenopus. Douglas W. Houston. 2018. [42].
  • Applying Tensile and Compressive Force to Xenopus Animal Cap Tissue. Georgina K. Goddard, Nawseen Tarannum, and Sarah Woolner. 2020. [43].
  • Microinjection of mRNAs and Oligonucleotides. Sally A. Moody. 2018. [44]
  • Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes. Samantha P. Jeschonek and Kimberly L. Mowry. 2018. [45]
  • Microinjection of DNA Constructs into Xenopus Embryos for Gene Misexpression and cis-Regulatory Module Analysis. Yuuri Yasuoka and Masanori Taira. 2019. [46]

'Omics

• • Transcriptomics and Proteomics Methods for Xenopus Embryos and Tissues. Michael J. Gilchrist, Gert Jan C. Veenstra, and Ken W.Y. Cho. 2020. ''Topic Introduction'' [47]
  • 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. [48]
  • INTACT Proteomics in Xenopus. Lauren Wasson, Nirav M. Amin, and Frank L. Conlon. 2019. [49]

Genomes, Chromosomes, DNA, Chromatin and Epigenetics

• • In Vitro Transcription Systems. Michael R. Green and Joseph Sambrook. [50]
  • ChIP-Sequencing in Xenopus Embryos. Saartje Hontelez, Ila van Kruijsbergen, and Gert Jan C. Veenstra. 2019. [51]
  • Chromatin Characterization in Xenopus laevis Cell-Free Egg Extracts and Embryos. Wei-Lin Wang, Takashi Onikubo, and David Shechter. 2019. [52].
  • Analysis of Chromatin Binding of Ectopically Expressed Proteins in Early Xenopus Embryos. Laura J.A. Hardwick and Anna Philpott. 2019. [53].
  • Analysis of Phosphorylation Status of Ectopically Expressed Proteins in Early Xenopus Embryos. Laura J.A. Hardwick and Anna Philpott. 2019. [54].
  • Assessing Ubiquitylation of Individual Proteins Using Xenopus Extract Systems. Gary S. McDowell and Anna Philpott. 2019. [55]
  • Generating a Three-Dimensional Genome from Xenopus with Hi-C. Ian K. Quigley and Sven Heinz. 2019. [56]
  • An RNA-Seq Protocol for Differential Expression Analysis. Nick D.L. Owens, Elena De Domenico, and Michael J. Gilchrist. 2019. [57]
  • DNase-seq to Study Chromatin Accessibility in Early Xenopus tropicalis Embryos. Jin Sun Cho, Ira L. Blitz, and Ken W.Y. Cho. 2019 [58]
  • Mapping Chromatin Features of Xenopus Embryos. George E. Gentsch and James C. Smith. 2019. [59]
  • Reconstituting Nuclear and Chromosome Dynamics Using Xenopus Extracts. Susannah Rankin. 2019 [60]
  • Extracts for Analysis of DNA Replication in a Nucleus-Free System. Justin Sparks and Johannes C. Walter. 2019. [61]
  • Endoplasmic Reticulum Network Formation with Xenopus Egg Extracts. Songyu Wang, Fabian B. Romano, and Tom A. Rapoport. 2019. [62].
  • Chromosome Cohesion and Condensation in Xenopus Egg Extracts. Eulália M.L. da Silva and Susannah Rankin. 2019. [63]

Endocrinology, Toxicology and Metamorphosis

• • Methods for Investigating the Larval Period and Metamorphosis in Xenopus. Daniel R. Buchholz and Yun-Bo Shi. ''Topic Introduction'' [64].
  • 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: [65].

Cell-free extracts, Cell-free systems

• • The Use of Cell-Free Xenopus Extracts to Investigate Cytoplasmic Events. Romain Gibeaux and Rebecca Heald. 2019.''Topic Introduction'' [66]
  • Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems. Gary S. McDowell and Anna Philpott. 2019. [67].
  • Special Considerations for Making Explants and Transplants with Xenopus tropicalis. 2019. Marilyn Fisher and Robert M. Grainger. [68]. Supplemental Material [69].
  • Filopodia-Like Structure Formation from Xenopus Egg Extracts. Helen M. Fox and Jennifer L. Gallop. 2019. [70].
  • Centromere and Kinetochore Assembly in Xenopus laevis Egg Extract. Julio C. Flores Servin and Aaron F. Straight. 2018. [71]
  • Dissecting Protein Complexes in Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts. Jae-Geun Song and Sabine Petry. 2018. [72]
  • Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts. Christopher Jenness, David J. Wynne, and Hironori Funabiki. 2018. [73]
  • Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts. Yinghui Mao. 2018 [74]
  • Chemical Screening Using Cell-Free Xenopus Egg Extract. Matthew R. Broadus and Ethan Lee. 2018. [75]
  • Robustly Cycling Xenopus laevis Cell-Free Extracts in Teflon Chambers. Jeremy B. Chang and James E. Ferrell Jr. [76]
  • Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts. John Oakey and Jesse C. Gatlin. 2018. [77]

Immunobiology

• • Raising Antibodies for Use in Xenopus. Maya Z. Piccinni and Matthew J. Guille. 2020. [78]
  • Purifying Antibodies Raised against Xenopus Peptides. Maya Z. Piccinni and Matthew J. Guille. 2020. [79]
  • Assessing the Immune Response When Raising Antibodies for Use in Xenopus. Maya Z. Piccinni and Matthew J. Guille. 2020. [80].
  • Lymphocyte Deficiency Induced by Sublethal Irradiation in Xenopus. Louise A. Rollins-Smith and Jacques Robert. 2019. [81].
  • Skin Grafting in Xenopus laevis: A Technique for Assessing Development and Immunological Disparity. Yumi Izutsu. 2019. [82]
  • Adoptive Transfer of Fluorescently Labeled Immune Cells in Xenopus. Kun Hyoe Rhoo and Jacques Robert. [83]
  • Assessing Antibody Responses to Pathogens or Model Antigens in Xenopus by Enzyme-Linked Immunosorbent Assay (ELISA). Francisco De Jesús Andino and Jacques Robert. 2019 [84]
  • In Vivo Assessment of Neural Precursor Cell Cycle Kinetics in the Amphibian Retina. Morgane Locker and Muriel Perron2019. [85], Supplemental Material [86]
  • Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts. Christopher Jenness, David J. Wynne, and Hironori Funabiki. 2018. [87]

Neurobiology and tissue regeneration

• • Ex Vivo Eye Tissue Culture Methods for Xenopus. Jonathan J. Henry, Kimberly J. Perry, and Paul W. Hamilton. 2019. [88].
  • Methods for Examining Lens Regeneration in Xenopus. Jonathan J. Henry, Kimberly J. Perry, and Paul W. Hamilton. 2019. [89]
  • Tracing Central Nervous System Axon Regeneration in Xenopus. Kurt M. Gibbs and Ben G. Szaro. 2018. [90]
  • Cell Transplantation as a Method to Investigate Spinal Cord Regeneration in Regenerative and Nonregenerative Xenopus Stages. Emilio E. Méndez-Olivos and Juan Larraín. 2018. [91]
  • Infrared Laser-Mediated Gene Induction at the Single-Cell Level in the Regenerating Tail of Xenopus laevis Tadpoles. Riho Hasugata, Shinichi Hayashi, Aiko Kawasumi-Kita, Joe Sakamoto, Yasuhiro Kamei, and Hitoshi Yokoyama. 2018. [92]
  • Rod-Specific Ablation Using the Nitroreductase/Metronidazole System to Investigate Regeneration in Xenopus. Reyna I. Martinez-De Luna and Michael E. Zuber. 2018. [93]
  • Studies of Limb Regeneration in Larval Xenopus. Anthony L. Mescher and Anton W. Neff. 2019. [94].
  • Bulk Electroporation-Mediated Gene Transfer into Xenopus Tadpole Brain. Cristina Sáenz de Miera, Ethan Parr, and Robert J. Denver. 2018. [95] . Supplemental Material [96].

Fate mapping, explants and transplants

• • Dissecting and Culturing Animal Cap Explants. Kevin S. Dingwell and James C. Smith. 2018. [97]
  • Einsteck Transplants. Hélène Cousin. 2019 [98]
  • Cleavage Blastomere Deletion and Transplantation to Test Cell Fate Commitment in Xenopus. Sally A. Moody. 2019. [99]
  • Cleavage Blastomere Explant Culture in Xenopus. Sally A. Moody. 2019. [100]
  • Lineage Tracing and Fate Mapping in Xenopus Embryos. Sally A. Moody. 2018. [101]

2019/2020 CSHL Recipes

• • Embryo Lysis Buffer (Xenopus). (Recipe 1) CSHLP. 2019 [102]
  • Xenopus Embryo Lysis Buffer. (Recipe 2). CSHLP. 2019 [103]
  • Lysis Buffer for Xenopus (recipe3) CSHLP. 2019. [104]
  • DNA Isolation Buffer. CSHLP. 2019 [105]
  • Lysis Buffer for Xenopus Hi-C. CSHLP 2019. [106]
  • RIPA Buffer for Xenopus. CSHLP 2019. [107]
  • Amphibian Serum-Free (ASF) Medium Supplemented with Fetal Bovine Serum (FBS). CSHL 2019. [108]
  • Xenopus Eye Culture Medium. CSHLP 2019. [109]
  • Marc's Modified Ringer's (MMR) for Xenopus (20×). CSHLP. 2019. [110]
  • Injection Buffer for Xenopus. CSHLP 2019. [111]
  • TE Buffer for Xenopus. CSHLP 2019. [112]
  • Proteinase K Buffer for Xenopus Oocytes. CSHLP 2018. [113]
  • PBT/PBT-Plus for Xenopus Oocytes. CSHL. 2018. [114]

Cold Spring Harbor Xenopus Protocols 2007. Edited by Hazel Sive

• • Housing and Feeding of Xenopus laevis - Sive et al. [115]
  • Inducing Ovulation in Xenopus laevis - Sive et al. [116]
  • Xenopus laevis In Vitro Fertilization and Natural Mating Methods - Sive et al. [117]
  • Egg Collection and In Vitro Fertilization of the Western Clawed Frog Xenopus tropicalis - Showell & Conlon [118]
  • Isolation of Xenopus Oocytes - Sive et al. [119]
  • Isolating Xenopus laevis Testes - Sive et al. [120]
  • Dejellying Xenopus laevis Embryos - Sive et al. [121]
  • Removing the Vitelline Membrane from Xenopus laevis Embryos - Sive et al. [122]
  • Microinjection of Xenopus Embryos - Sive et al. [123]
  • Defolliculation of Xenopus Oocytes - Sive et al. [124]
  • Microinjection of Xenopus Oocytes - Sive et al. [125]
  • Animal Cap Isolation from Xenopus laevis - Sive et al. [126]
  • Xenopus laevis Keller Explants - Sive et al. [127]
  • Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes - Sive et al. [128]
  • Calibration of the Injection Volume for Microinjection of Xenopus Oocytes and Embryos - Sive et al. [129]
  • Isolation of DNA from Red Blood Cells in Xenopus - Sive et al. [130]
  • Investigating Morphogenesis in Xenopus Embryos: Imaging Strategies, Processing, and Analysis - Kim & Davidson [131]
  • Low-Magnification Live Imaging of Xenopus Embryos for Cell and Developmental Biology - Wallingford [132]
  • High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology - Keiserman et al. [133]
  • Preparation of Fixed Xenopus Embryos for Confocal Imaging - Wallingford [134]
  • Whole-Mount Fluorescence Immunocytochemistry on Xenopus Embryos - Lee et al. [135]
  • Generation of Transgenic Xenopus laevis - Kroll & Amaya - [136] [137] [138]
  • In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus - Ruthazer et al. [139]
  • Photoconversion for Tracking the Dynamics of Cell Movement in Xenopus laevis Embryos - Chernet et al. [140]
  • Single-Cell Electroporation in Xenopus - Liu & Haas [141]
  • Imaging Axon Pathfinding in Xenopus In Vivo - Leung & Holt [142]
  • A Versatile Protocol for mRNA Electroporation of Xenopus laevis Embryos - Chernet & Levin [143]

General Research Protocols

Animal Husbandry

• obtain frogs and frog housing equipment [144]
• Xenopus Express links [145] - Xenopus Express husbandry [146]
• 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). [147]
• A method for generating transgenic frog embryos. Ishibashi S, Kroll KL, Amaya E. Methods Mol Biol. 2008;461:447-66. [148]
• A method for generating transgenic frog embryos. Amaya E, Kroll KL. Methods Mol Biol. 1999;97:393-414. [149]

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. [150] [151]
  • Akkers RC, Jacobi UG, Veenstra GJ.
• Chromatin immunoprecipitation in early Xenopus laevis embryos., Dev Dyn. 2009 Jun;238(6):1422-32. [152] [153]
  • 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)

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)