In situ characterization of genetically targeted (green fluorescent) single cells and their microenvironment in an adoptive host

Stable expression of transgene-encoded enhanced green fluorescence protein (eGFP) was used as a sensitive and specific marker to detect in situ donor cells engrafted into different tissues of adoptive hosts. eGFP(+) lymphoid or myeloid cells leg, CD4(+) T cells or bone marrow-derived dendritic cells ) from eGFP-transgenic C57BL/6 donor mice were injected into congenic, immu nodeficient RAG1(-/-) C57/BL6 hosts. eGFP(+) cells were detected in the ado ptive host from 2 days to 4 weeks after transfer using an optimized method of fixed cryopreservation to process the tissue. This allowed the simple, s ensitive, and specific detection of eGFP(+) donor cells in histological sec tions of transplanted hosts. We further demonstrate that this technique can be combined with other established labeling methods such as 1) immunofluor escent labeling to characterize the host cells interacting with engrafted c ells and to determine the phenotype of the engrafted cells in situ; 2) term inal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining to detect apoptotic death of engrafted and autochthonous cell populations; and 3) fluorescent antibody labeling of incorporated bromodeoxyuridine to m easure the fraction of proliferating cells in the graft.