Scott N. Mueller
  • Faculty, Microbiology and Immunology, The University of Melbourne, Australia
Research fields
  • Immunology
Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes
Authors:  Yannick O. Alexandre and Scott N Mueller, date: 08/20/2017, view: 9934, Q&A: 0
Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC) as PNAd+ BEC that form LN high endothelial venules (HEV). This method can be applied to examine LN stromal cell responses during inflammatory events induced by infections or immunologic adjuvants and to subset most leukocytes found in LN.
Skin TRITC Painting to Track Dendritic Cells Migrating to the Lymph Nodes
Authors:  Jyh Liang Hor and Scott N. Mueller, date: 09/20/2016, view: 8723, Q&A: 0
Our protocol describes a simple method that allows tracking of dendritic cells (DC) migration from the flank skin to draining lymph nodes (LN) using a red fluorescent dye tetramethylrhodamine-5-isothiocyanate (TRITC). TRITC is a photostable dye that readily labels cells including DC in the skin and can survive repeated exposure of two-photon laser excitation for prolonged imaging duration. This method can be combined with various fluorescent antibody labels or transgenic mouse strains (such as CD11c-EYFP) to visualize distinct DC populations simultaneously.
Imaging Thick Lymph Node Tissue Sections
Authors:  Jyh Liang Hor and Scott N. Mueller, date: 09/20/2016, view: 12425, Q&A: 0
Our protocol describes a simple procedure for imaging thick lymph node sections by 2-photon microscopy. Lymph nodes are sectioned using a vibratome (vibrating microtome) to produce slices of tissue that can then be stained with fluorescently labeled antibodies. The thick tissue sections (150-200 μm depth) allow for the detection of cell clustering that is typically under-represented in thin sections (10-20 μm) used for conventional confocal microscopy. Application of 2-photon microscopy facilitates imaging through the thick volume of the vibratome sections. In combination with automated image processing software, a thick lymph node cross-section image also facilitates quantitation of cellular events within a relatively large area of the tissue, thus providing a clearer picture on the spatial distribution of cellular events of interest (e.g., T cell clustering). This method can also readily be applied to other tissues, such as the spleen or skin.
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