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    Protocols in Current Issue
    Culture and Nucleofection of Postnatal Day 7 Cortical and Cerebellar Mouse Astroglial Cells
    Authors:  Malek Chouchane and Marcos Romualdo Costa, date: 02/05/2018, view: 151, Q&A: 0
    Lineage reprogramming of astroglial cells isolated from different brain regions leads to the generation of different neuronal subtypes. This protocol describes the isolation and culture of neocortical and cerebellar astrocytes from postnatal mice. ...
    Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae
    Authors:  Liang-Chun Wang, Qian Yu, Daniel C. Stein and Wenxia Song, date: 02/05/2018, view: 107, Q&A: 0
    Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually ...
    Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting
    To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the ...
    Ex vivo Analysis of Lipolysis in Human Subcutaneous Adipose Tissue Explants
    Authors:  Pauline Decaunes, Anne Bouloumié, Mikael Ryden and Jean Galitzky, date: 02/05/2018, view: 76, Q&A: 0
    Most studies of human adipose tissue (AT) metabolism and functionality have been performed in vitro on isolated mature adipocyte or in situ using the microdialysis technique (Lafontan, 2012). However, these approaches have several ...
    Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans
    Authors:  Julie M. Huynh, Hope Dang and Hanna Fares, date: 02/05/2018, view: 114, Q&A: 0
    Assays have been developed to study trafficking in various tissues of Caenorhabditis elegans. Adult C. elegans intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the ...
    Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana
    Authors:  Susumu Uehara, Yasuko Ito-Inaba and Takehito Inaba, date: 02/05/2018, view: 84, Q&A: 0
    In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific ...
    Analysis of Chromosome Condensation/Decondensation During Mitosis by EdU Incorporation in Nigella damascena L. Seedling Roots
    Author:  Eugene V. Sheval, date: 02/05/2018, view: 90, Q&A: 0
    To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in ...
    Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells
    The uptake and trafficking of cell surface receptors can be monitored by a technique called ‘antibody-feeding’ which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional ...
    Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging
    Authors:  Samuel Petshow and Marcel Wehrli, date: 02/05/2018, view: 136, Q&A: 0
    The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a ...
    Medaka-microinjection with an Upright Microscope
    Authors:  Yu Murakami and Masato Kinoshita, date: 02/05/2018, view: 115, Q&A: 0
    We described a simple method for microinjecting DNA/RNA/Protein solutions into medaka eggs under an upright microscope. Medaka is an excellent vertebrate model for reverse genetics, because of its daily spawning, short generation time, and egg ...
    Culture and Nucleofection of Postnatal Day 7 Cortical and Cerebellar Mouse Astroglial Cells
    Authors:  Malek Chouchane and Marcos Romualdo Costa, date: 02/05/2018, view: 151, Q&A: 0
    [Abstract] Lineage reprogramming of astroglial cells isolated from different brain regions leads to the generation of different neuronal subtypes. This protocol describes the isolation and culture of neocortical and cerebellar astrocytes from postnatal mice. We also present a comprehensive description of the main steps towards successful gene delivery in ...
    Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae
    Authors:  Liang-Chun Wang, Qian Yu, Daniel C. Stein and Wenxia Song, date: 02/05/2018, view: 107, Q&A: 0
    [Abstract] Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female ...
    Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting
    [Abstract] To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since N. meningitidis niche is restricted ...
    Ex vivo Analysis of Lipolysis in Human Subcutaneous Adipose Tissue Explants
    Authors:  Pauline Decaunes, Anne Bouloumié, Mikael Ryden and Jean Galitzky, date: 02/05/2018, view: 76, Q&A: 0
    [Abstract] Most studies of human adipose tissue (AT) metabolism and functionality have been performed in vitro on isolated mature adipocyte or in situ using the microdialysis technique (Lafontan, 2012). However, these approaches have several limitations. The use of mature isolated adipocytes is limiting as adipocytes are not in their ...
    Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans
    Authors:  Julie M. Huynh, Hope Dang and Hanna Fares, date: 02/05/2018, view: 114, Q&A: 0
    [Abstract] Assays have been developed to study trafficking in various tissues of Caenorhabditis elegans. Adult C. elegans intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the endocytic pathway using live imaging techniques. However, the presence of auto-fluorescent gut granules ...
    Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana
    Authors:  Susumu Uehara, Yasuko Ito-Inaba and Takehito Inaba, date: 02/05/2018, view: 84, Q&A: 0
    [Abstract] In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide ...
    Analysis of Chromosome Condensation/Decondensation During Mitosis by EdU Incorporation in Nigella damascena L. Seedling Roots
    Author:  Eugene V. Sheval, date: 02/05/2018, view: 90, Q&A: 0
    [Abstract] To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in plant chromosomes, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU), which ...
    Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells
    [Abstract] The uptake and trafficking of cell surface receptors can be monitored by a technique called ‘antibody-feeding’ which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on ...
    Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging
    Authors:  Samuel Petshow and Marcel Wehrli, date: 02/05/2018, view: 136, Q&A: 0
    [Abstract] The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D ...
    Medaka-microinjection with an Upright Microscope
    Authors:  Yu Murakami and Masato Kinoshita, date: 02/05/2018, view: 115, Q&A: 0
    [Abstract] We described a simple method for microinjecting DNA/RNA/Protein solutions into medaka eggs under an upright microscope. Medaka is an excellent vertebrate model for reverse genetics, because of its daily spawning, short generation time, and egg transparency. These features enable us to efficiently perform functional genomic analyses of transgenic ...