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  • 1.
    Andersson, Christopher
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Gripenland, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections2015In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 10, no 8, p. 1155-1164Article in journal (Refereed)
    Abstract [en]

    Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes <4 weeks to complete, with the infection part taking only 3 d. We discuss the appropriate controls to use and potential adjustments needed for adapting the protocol for other microbial pathogens.

  • 2.
    Boutté, Yohann
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Men, Shuzhen
    Grebe, Markus
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Fluorescent in situ visualization of sterols in Arabidopsis roots2011In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 6, no 4, p. 446-456Article in journal (Refereed)
    Abstract [en]

    Sterols are eukaryotic membrane components with crucial roles in diverse cellular processes. Elucidation of sterol function relies on development of tools for in situ sterol visualization. Here we describe protocols for in situ sterol localization in Arabidopsis thaliana root cells, using filipin as a specific probe for detection of fluorescent filipin-sterol complexes. Currently, filipin is the only established tool for sterol visualization in plants. Filipin labeling can be performed on aldehyde-fixed samples, largely preserving fluorescent proteins and being compatible with immunocytochemistry. Filipin can also be applied for probing live cells, taking into account the fact that it inhibits sterol-dependent endocytosis. The experimental procedures described are designed for fluorescence detection by confocal laser-scanning microscopy with excitation of filipin-sterol complexes at 364 nm. The protocols require 1 d for sterol covisualization with fluorescent proteins in fixed or live roots and 2 d for immunocytochemistry on whole-mount roots.

  • 3. Felten, Judith
    et al.
    Hall, Hardy
    Jaumot, Joaquim
    Tauler, Roma
    de Juan, Anna
    Gorzsas, Andras
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vibrational spectroscopic image analysis of biological material using multivariate curve resolution-alternating least squares (MCR-ALS)2015In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 10, no 2, p. 217-240Article in journal (Refereed)
    Abstract [en]

    Raman and Fourier transform IR (FTIR) microspectroscopic images of biological material (tissue sections) contain detailed information about their chemical composition. The challenge lies in identifying changes in chemical composition, as well as locating and assigning these changes to different conditions (pathology, anatomy, environmental or genetic factors). Multivariate data analysis techniques are ideal for decrypting such information from the data. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and unmixing of pixel spectra into their contributing pure components by multivariate curve resolution-alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile in order to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually <30-60 min per image), and they are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or heat maps of single-band intensities. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k-means clustering) is possible.

  • 4. Hmelo, Laura R.
    et al.
    Borlee, Bradley R.
    Almblad, Henrik
    Love, Michelle E.
    Randall, Trevor E.
    Tseng, Boo Shan
    Lin, Chuyang
    Irie, Yasuhiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Storek, Kelly M.
    Yang, Jaeun Jane
    Siehnel, Richard J.
    Howell, P. Lynne
    Singh, Pradeep K.
    Tolker-Nielsen, Tim
    Parsek, Matthew R.
    Schweizer, Herbert P.
    Harrison, Joe J.
    Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange2015In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 10, no 11, p. 1820-1841Article in journal (Refereed)
    Abstract [en]

    Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNANA. The entire procedure requires similar to 2 weeks.

  • 5.
    Norregaard, Kamilla
    et al.
    The Niels Bohr Institute.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.
    Nielsen, Peter
    Department of Cellular and Molecular Medicine, Faculty of Health and Sciences, University of Copenhagen, Copenhagen, Denmark.
    Brown, Stanley
    Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark .
    Oddershede, Lene
    Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.
    Tethered particle analysis of supercoiled circular DNA using PNA handles2014In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 9, no 9, p. 2206-2223Article in journal (Refereed)
  • 6.
    Obernosterer, Gregor
    et al.
    Austrian Academy of Science.
    Martinez, Javier
    Austrian Academy of Science.
    Alenius, Mattias
    IMP, Vienna.
    Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections2007In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 2, no 6, p. 1508-1514Article in journal (Refereed)
    Abstract [en]

    Here we describe a method for sensitive and specific histological detection of microRNAs (miRNAs) by in situ hybridization. The protocol focuses on the use of locked nucleic acids (LNAs), which are bi-cyclic RNA analogs that allow a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection. The protocol is optimized for cryosections in order to study the spatial and temporal expression of miRNAs with high sensitivity and resolution. We detail how to construct probes, set up and conduct an LNA in situ hybridization experiment. In addition, we discuss alternative colorimetric strategies that can be used to effectively detect and visualize miRNAs including double staining with other markers. Setting up and conducting the in situ experiment is estimated to take similar to 1 week, assuming that all the component parts are readily available.

1 - 6 of 6
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