Methods in Molecular Biology Volume 411, 2007, pp 145-154

Green Fluorescent Protein as a Tracer in Chimeric Tissues

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Green fluorescent protein (GFP) and its variants, small, highly soluble proteins, are routinely used as reporters for patterns of gene expression and the origin of cells in transplantation experiments. When not linked as fusion proteins to other polypeptides, they distribute rapidly in the cytoplasm of a given cell, thus allowing real-time observations on living material. For histological analysis, previous bath fixation of whole organs or tissues seemed obligatory, because, during drop fixation of sections, GFP rapidly leaks from cells whose membrane has been damaged by freezing and/or sectioning. The fluorescence of GFP and its derivatives is retained upon fixation, but most enzyme and antigenic activities of interest will be lost in the whole sample as a consequence of form aldehyde (FA) fixation. We have therefore developed an alternative method to fix GFP in frozen tissue sections by FA vapor. This method prevents leakage and redistribution of GFP and allows any cytochemical method to be applied to unfixed adjacent serial sections.