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Cold Spring Harbor Protocols

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https://www.readbyqxmd.com/read/29208644/filopodia-like-structure-formation-from-xenopus-egg-extracts
#1
Helen M Fox, Jennifer L Gallop
The actin cytoskeleton comprises many different architectures of filaments, including branched networks, parallel bundles and antiparallel fibers. A current challenge is to elucidate how the diverse array of actin regulators, which controls the growth, assembly and turnover of actin filaments, is used to orchestrate cytoskeletal organization and in turn cell shape and movement. Long observed to assemble at cell membranes, actin in Xenopus egg extracts recapitulates membrane-triggered assembly at specific lipid and membrane environments...
December 5, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29208643/heterologous-protein-expression-in-the-xenopus-oocyte
#2
Jonathan S Marchant
The Xenopus oocyte is a specialized single cell of colossal size (>1 mm diameter) that is highly amenable for microinjection and a stalwart model for heterologous expression. Oocytes are easily obtainable, robust in vitro, and faithfully express injected constructs. Their large size translational capacity provides a huge canvas for observing and recording integrated cellular responses-from studies of single molecules within single cells to medium-throughput drug-screening applications. Most eukaryotic promoters suffice for Xenopus expression, and the oocyte can functionally express proteins from many diverse organisms...
December 5, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29192091/whole-mount-immunocytochemistry-in-xenopus
#3
Michael W Klymkowsky
To visualize the effects of experimental perturbations on normal cellular behavior, morphology, and intracellular organization, we use a simple whole-mount immunocytochemical method with Xenopus oocytes, explants, or embryos. This method is applicable to a wide range of systems, including human-induced pluripotent stem cell-derived organoids, and can be used with both chromogenic (horseradish peroxidase/diaminobenzidine) and fluorescent imaging.
November 30, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29084864/whole-mount-in-situ-hybridization-of-xenopus-embryos
#4
Jean-Pierre Saint-Jeannet
Historically, techniques to analyze the localized distribution of mRNAs during development were performed on sectioned embryos using radioactively labeled riboprobes. The processing of the tissues and the use of emulsion autoradiography were laborious and time-consuming, leading to the development of more direct approaches. The nonradioactive whole-mount in situ hybridization method was first introduced in Drosophila embryos, and later adapted to Xenopus embryos for abundant transcripts such as muscle actin...
October 30, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29084863/in-vitro-induction-of-xenopus-embryonic-organs-using-animal-cap-cells
#5
Takashi Ariizumi, Tatsuo Michiue, Makoto Asashima
The animal cap-the presumptive ectoderm of the blastula embryo-can differentiate into a variety of tissues belonging to the three germ layers following exposure to specific inducers. The "animal cap assay" was devised based on the pluripotency of presumptive ectodermal cells and enabled many important discoveries in the field of embryonic induction and cell differentiation. Using this system, investigators can test multiple factors in solution simultaneously to determine their inducing activities qualitatively, quantitatively, and synergistically...
October 30, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29084862/elicitation-of-xenopus-laevis-tadpole-and-adult-frog-peritoneal-leukocytes
#6
Leon Grayfer
Peritoneal lavage of Xenopus laevis tadpoles and adult frogs is a reliable way of isolating resident and/or recruited innate immune populations. This protocol details the isolation of tadpole and adult amphibian (Xenopus laevis) peritoneal leukocytes. The isolated cells are comprised predominantly of innate immune populations and chiefly of mononuclear and polymorphonuclear granulocytes. As described here, these cells are typically elicited by peritoneal injections of animals with heat-killed Escherichia coli, causing peritoneal accumulation of inflammatory cell populations, which are then isolated from the stimulated animals by lavage...
October 30, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29196601/immunoprecipitation
#7
James DeCaprio, Thomas O Kohl
This immunoprecipitation protocol details individual steps for the enrichment and purification process of specific proteins from a complex cell lysate using an antibody bound to a solid matrix. Purified antigen(s) can be eluted by various methods, and the resultant protein target can be analyzed and/or identified by numerous assays, including the enzyme-linked immunosorbent assay (ELISA), western blotting, or mass spectrometry.
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29196600/detergent-lysis-of-animal-tissues-for-immunoprecipitation
#8
James DeCaprio, Thomas O Kohl
This protocol details protein extraction from mouse tissues for immunoprecipitation purposes and has been applied for the performance of large-scale immunoprecipitations of target proteins from various tissues for the identification of associated proteins by mass spectroscopy. The key factors in performing a successful immunoprecipitation directly relate to the abundance of target protein in a particular tissue type and whether or not the embryonic, newborn, or adult mouse-derived tissues contain fibrous and other insoluble material...
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29196599/detergent-lysis-of-tissue-culture-cells-for-immunoprecipitation
#9
James DeCaprio, Thomas O Kohl
This protocol describes the lysis of tissue culture cells for the solubilization of proteins of interest for immunoprecipitation. Upon collection of cells by centrifugation and depending on the use of either Tris- or phosphate-based cell lysis buffers, cells are rinsed, respectively, in either TBS or PBS before lysis. If possible, the pH of the Wash buffer should match that of the Lysis buffer. Adherent cells can be directly lysed on the plate. This is particularly useful upon lysis encompassing mild nonionic detergents, leaving the cytoskeleton intact...
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29196598/preparation-of-dna-from-embryonic-stem-cells-or-other-cultured-cells
#10
Richard Behringer, Marina Gertsenstein, Kristina Vintersten Nagy, Andras Nagy
Characterization of embryonic stem (ES) cell-mediated genome alterations, including random insertional transgenesis, gene trapping, gene targeting, and site-specific recombinase-mediated changes, is performed mostly at the ES cell level, before the introduction of these alterations into a mouse. A detailed characterization requires a larger amount of DNA than is required for the initial detection of the candidates for the desired alteration. This protocol describes the preparation of DNA from a 10-cm tissue culture plate...
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29196597/testing-serum-batches-for-mouse-embryonic-stem-cell-culture
#11
Richard Behringer, Marina Gertsenstein, Kristina Vintersten Nagy, Andras Nagy
The variability in embryonic stem (ES) cell culture is due primarily to serum. Serum is typically produced in large batches from many animals. However, samples may differ depending on the age and diet of the animals, the country of origin, and other factors creating lot-to-lot variations. Some vendors test FBS lots for compatibility with ES cell culture. Many laboratories prefer to test serum batches themselves to identify the lot giving optimal growth. In this protocol, small quantities of specific serum batches are obtained from different suppliers and tested for their ability to support ES cells in an undifferentiated state...
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29196596/corrigendum-methods-to-synthesize-large-dna-fragments-for-a-synthetic-yeast-genome
#12
Yizhi Cai, Junbiao Dai
No abstract text is available yet for this article.
December 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093209/characterizing-antibodies
#13
Frances Weis-Garcia, Robert H Carnahan
Perhaps because they are such commonly used tools, many researchers view antibodies one-dimensionally: Antibody Y binds antigen X. Although few techniques require a comprehensive understanding of any particular antibody's characteristics, well-executed experiments do require a basic appreciation of what is known and, equally as important, what is not known about the antibody being used. Ignorance of the relevant antibody characteristics critical for a particular assay can easily lead to loss of precious resources (time, money, and limiting amounts of sample) and, in worst-case scenarios, erroneous conclusions...
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093208/harvesting-tissue-from-immunized-mice-rats-and-hamsters
#14
Edward A Greenfield
Immunologically active organs such as the spleen and lymph nodes are rich sources of antibodies; they are also major sites in the body where antibody-producing B cells accumulate. The lymph nodes, in particular, can be targeted when deciding where to inject an animal with antigen. Once an immunized animal has developed a sufficient serum antibody titer, these organs can be harvested for hybridoma fusion and monoclonal antibody production.
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093207/sampling-and-preparation-of-mouse-and-rat-serum
#15
Edward A Greenfield
After immunization, samples of serum are taken to check the production of specific antibodies. Serum is collected from clotted blood or plasma without the fibrinogen and other clotting factors. Serum contains fewer proteins than plasma and is preferred for use in many clinical assays because some of the proteins in plasma or the addition of anticoagulants can interfere with the test results. Methods for obtaining blood samples and preparing serum are described here. Blood collection should not exceed 7%-9% of the animal's total blood volume or 1% of total body weight every 2-4 wk...
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093206/in-vivo-transfection-of-naked-dna-into-xenopus-tadpole-tail-muscle
#16
Lindsey Marshall, Fabrice Girardot, Barbara A Demeneix, Laurent Coen
In vivo gene transfer systems are important to study foreign gene expression and promoter regulation in an organism, with the benefit of exploring this in an integrated environment. Direct injection of plasmids encoding exogenous promoters and genes into muscle has numerous advantages: the protocol is easy, efficient, and shows time-persistent plasmid expression in transfected muscular cells. After injecting naked-DNA plasmids into tadpole tail muscle, transgene expression is strong, reproducible, and correlates with the amount of DNA injected...
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093205/xenopus-tadpole-tissue-harvest
#17
Matthew D Patmann, Leena H Shewade, Katelin A Schneider, Daniel R Buchholz
The procedures described here apply to Xenopus tadpoles from the beginning of feeding through the major changes of metamorphosis and are appropriate for downstream postoperative snap freezing for molecular analysis, fixation for histological analysis, and sterile organ culture. To the uninitiated, the most difficult aspects of tadpole tissue dissections are likely knowing the appearance and location of organs, and the difficulty manipulating and holding tadpoles in place to carry out the oftentimes fine and precise dissections...
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093204/in-vitro-fertilization-in-mice
#18
Robert Taft
In vitro fertilization (IVF) involves fertilization of mature oocytes with capacitated sperm in a tissue culture dish. This technique can generate large numbers of cleavage-stage embryos without using a significant number of single-caged stud males for mating. In addition, sperm penetration is synchronous during incubation with mature oocytes, leading to synchronous development, unlike fertilization in vivo, when natural ovulation is usually spread over time. These embryos can be used for a variety of applications including rapid strain expansion and generation of age-matched experimental cohorts as well as cryopreservation at the two-cell stage (speed cryo)...
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093203/mouse-sperm-cryopreservation-using-cryoprotectant-containing-monothioglycerol-mtg
#19
Robert Taft
Significant progress has been made in sperm cryopreservation. It is now possible to achieve more consistent fertilization rates using in vitro fertilization (IVF) with cryorecovered sperm. Including the antioxidant monothioglycerol (MTG) in the cryoprotectant may increase the fertilization rate. This protocol has been successfully used at The Jackson Laboratory since 2007 to cryopreserve >5000 strains on many genetic backgrounds. Although it is possible to recover motile sperm from 2-yr-old mice, sperm quality often begins to decline by 6 mo of age...
November 1, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29093202/isotype-determination-of-rodent-derived-monoclonal-antibodies-using-sandwich-elisa
#20
Frances Weis-Garcia, Robert H Carnahan
For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody...
November 1, 2017: Cold Spring Harbor Protocols
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