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See detailLaser-induced choroidal neovascularization model to study age-related macular degeneration in mice.
LAMBERT, Vincent ULg; Lecomte, Julie ULg; Hansen, Sylvain ULg et al

in Nature Protocols (2013), 8(11), 2197-2211

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model ... [more ▼]

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7-14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature. [less ▲]

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See detailAgrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.
Bull, S. E.; Owiti, J. A.; Niklaus, M. et al

in Nature protocols (2009), 4(12), 1845-54

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and ... [more ▼]

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop. [less ▲]

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See detailThe lymphatic ring assay: a 3D-culture model of lymphangiogenesis.
Bruyere, Françoise ULg; Melen-Lamalle, Laurence ULg; Berndt, Sarah ULg et al

in Nature Protocols (2008)

Lymphangiogenesis, the formation of new lymphatic vessels, is associated to numerous pathologies1 and understanding the molecular and cellular basis of this complex process is essential for the ... [more ▼]

Lymphangiogenesis, the formation of new lymphatic vessels, is associated to numerous pathologies1 and understanding the molecular and cellular basis of this complex process is essential for the development of novel therapeutic strategies. Studies on lymphangiogenesis have been hampered by difficulties in culturing lymphatic capillaries as three-dimensional (3D) structures in vitro that mimic the in vivo features of lymphatic vessels and lymphangiogenesis. The lymphatic ring assay described here phenocopies the different steps of lymphangiogenesis, including the spreading from a preexisting vessel, cell proliferation, migration and differentiation into capillaries. It consists on the adaptation of the aortic ring assay that has proved to be useful to investigate the molecular basis of angiogenesis2-4. The lymphatic ring model is an ideal assay for testing the activity of lymphangiogenic agonists or antagonists. The absence of inflammatory cells allows a simple interpretation of results and the determination of direct effects of compounds on lymphatic endothelial cell properties. Another advantage of the lymphatic ring assay is that cell outgrowing are primary cells which have not been modified by repeated passages or immortalization. This culture model bridges the gap between in vitro and in vivo studies and allows genetic analysis by using thoracic ducts from genetically modified mice. [less ▲]

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See detailA protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.
Thiry, Marc ULg; Lamaye, Françoise ULg; Thelen, Nicolas ULg et al

in Nature Protocols (2008), 3(12), 1997-2004

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living ... [more ▼]

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d. [less ▲]

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See detailAtomic force microscopy of supported lipid bilayers.
Mingeot-Leclercq, Marie*-Paule; Deleu, Magali ULg; Brasseur, Robert ULg et al

in Nature Protocols (2008), 3(10), 1654-9

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy ... [more ▼]

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h. [less ▲]

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See detailPatch-clamp recording from mossy fiber terminals in hippocampal slices
Bischofberger, J; Engel, Dominique ULg; Li, L et al

in Nature Protocols (2006), 1(4), 2075-81

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