A method for fixing and paraffin embedding tissue to retain the natural fluorescence of reporter proteins.
Green fluorescent protein (GFP) and its derivatives are routinely employed as surrogate markers for gene expression and lineage tracing in genetically engineered mice. Tissues from these mice are commonly formalin fixed and paraffin embedded (FFPE) for histological studies. However, this results in inactivation of the natural fluorescence of these proteins, requiring their detection by immunological techniques. Here we present an ethanol fixation protocol that allows for the direct visualization of the natural fluorescence of reporter proteins while maintaining excellent tissue histology. We demonstrate the utility of this method for visualizing green and red fluorescent proteins in a wide range of murine tissues using both cytoplasmic and membrane-localized fluorescent reporter proteins. Tissues fixed by this method also allow for immunohistochemical studies, providing a single method to visualize the natural fluorescence of reporter proteins with subsequent detection of cellular proteins.
Department of Surgery and the Andrew L Warshaw, MD, Institute for Pancreatic Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Akifumi
A
Nakagawa
Department of Surgery and the Andrew L Warshaw, MD, Institute for Pancreatic Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Kate Von
KV
Alt
Department of Surgery and the Andrew L Warshaw, MD, Institute for Pancreatic Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Keith D
KD
Lillemoe
Department of Surgery and the Andrew L Warshaw, MD, Institute for Pancreatic Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Carlos Fernández-Del
CF
Castillo
Department of Surgery and the Andrew L Warshaw, MD, Institute for Pancreatic Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Andrew L
AL
Warshaw
Department of Surgery and the Andrew L Warshaw, MD, Institute for Pancreatic Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Andrew S
AS
Liss
D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008822:Mice, Transgenic; D016612:Paraffin Embedding / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods*; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein
FFPE; GFP; natural fluorescence