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Direct detection of green fluorescent protein in histological preparations of recombinant vaccinia virus in mice (1050.9)
Author(s) -
Lyons Cassandra
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1050.9
Subject(s) - green fluorescent protein , virology , virus , recombinant dna , biology , staining , immunohistochemistry , vaccinia , microbiology and biotechnology , ex vivo , recombinant virus , poxviridae , in vivo , gene , immunology , biochemistry , genetics
Green fluorescent protein (GFP)‐expressing recombinant viruses have proven to be invaluable tools for the in‐vivo monitoring of viral kinetics and disease progression in infectious disease models. Histopathological evaluation of the ex‐vivo tissues by way of immunohistochemical (IHC) staining for viral antigens further assists in the characterization of disease pathology, but can be insensitive, nonspecific, and time consuming. In this study, we determine if the direct detection of GFP signal provides a suitable alternative to IHC staining in GFP‐expressing tissues. Fixation and histological processing protocols were developed in a GFP‐expressing 293T cells and then optimized and validated in a GFP‐recombinant rat model. The optimized protocol was then applied to GFP‐recombinant cowpox virus (VACV‐TK‐GFP) infected mouse lungs in addition to lungs infected with a wild‐type virus strain (VACV‐WR). While animals infected with both virus strains manifested lesions reactive to anti‐Vaccinia IHC, GFP fluorescence was only detected in lesions from animals infected with the recombinant virus strain. The method was further validated through IHC staining of both virus strains with anti‐GFP antibodies, which were only reactive in tissues infected with VACV‐TK‐GFP. Digital alignment of sections showed a strong correlation between GFP fluorescence and IHC staining for GFP and viral proteins suggesting direct‐detection of fluorescent proteins is a promising alternative to traditional methods.