Fluorescence in-situ hybrydisation and immunofluorescence for characterizing cell transplants
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Aims. Extend the utility of fluorescence in-situ hybridisation (FISH) targeting specific DNA repeat sequences, in combination with multiple labelling immunofluorescence for a range of marker proteins to analyse cell grafts within the CNS. Background. Cell transplantation into animal models is used in a broad spectrum of research, with a critical aspect of these studies being the identification and characterization of implanted cells. Such analysis becomes challenging in studies involving large numbers of grafted cells following proliferation and/or differentiation within host tissue. Furthermore, the use of reporter expression and fluorescent cell tracers is limited due to potential changes in gene expression patterns or dispersal of cell tracers. Here we distinguish and characterise cells based on their intrinsic molecular properties in several animal models including rat, mouse and guinea-pig . Methods. We utilise sectioning and labelling protocols maximising preservation of molecular and anatomical properties of tissues; the tissue is impregnated with polyethylene glycol and sectioned at room temperature. Biotinylated oligonucleotide sequences are hybridised onto specimens, followed by incubation in a range of antisera targeting diverse antigens including structural proteins, neurotransmitters, enzymes and membrane associated proteins and visualised in fine optical sections using wide-field fluorescence microscopy. Conclusions FISH for cell specific DNA is an accurate method of lineage identification, and combined with multiple labelling immunofluorescence, offers a robust method for cell characterisation. 1. Robinson, A. et al (2005) Survival and engraftment of mouse embryonic stem cell-derived implants in the guinea pig brain. Neurosci Res, 53(2): 161-168.
International Brain Research Organisation / Australian Neuroscience Society Conference
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