Visualisation of Lymphoid Organ Development
This chapter provides information on imaging tools that can be employed to visualise and study lymphoid organ development. We focus on the use of genetically modified mouse models that take advantage of fluorescent protein expression in discrete cell populations, thus allowing live cell imaging during lymphoid organogenesis. We discuss approaches that allow characterisation of the cell types involved in the formation of lymphoid organs, including (i) functional assays in explant organ cultures and (ii) high-resolution whole-mount immunostaining methods, which are useful for the characterisation of specific cell populations in the context of the whole developing organ.
Key wordsLymphoid organogenesis genetically modified animals fluorescent proteins live cell imaging time-lapse microscopy stereo fluorescent microscopy confocal laser scanning microscopy imaging
Work in this chapter was funded by the Medical Research Council (MRC), UK. We wish to thank Vassilis Pachnis for helpful discussion; T. Norton and K. Williams for technical assistance. We also wish to thank Prof. D. Vestweber for kindly providing endomucin antibody. H.V.-F. and K.F. were supported by a grant from the European Union: Molecular Imaging LSHG-CT-2003-503259
Supplementary Video 1. Time-lapse video showing mobility of GFP cells in the wall of the gut (low magnification). This video shows a time-lapse sequence of E15.5 intestines. Time-lapse images were taken for 90 min.
Supplementary Video 2. Three-dimensional reconstitution of an embryo section of E13.5. This sample was immunostained with anti-GFP (green) and the embryo structure is depicted in grey color. This video shows the developing lymph nodes and thymus. Magnification ×10.
Supplementary Video 3. Three-dimensional reconstitution of an adult thymus section. This sample was immunostained with anti-endomucin (red). This video shows the vessel and capillary endomucin-positive network within the thymus. Magnification ×40.
Paraformaldehyde fixation times vary according to size of tissues or organs. As guidance, E15.5 intestine will require 15 min fixation at room temperature. These conditions may however vary according to the tissue and antibodies being used.
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