Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation.
Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.
Trotman
Winifred
W
Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Beckett
Travis
T
Goncz
Kaarin K
KK
Beatty
Barbara G
BG
Weiss
Daniel J
DJ
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D016026:Bone Marrow Transplantation; D020223:Chromosome Painting; D005260:Female; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D066298:In Vitro Techniques; D008168:Lung / Q000737:chemistry* / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D014998:Y Chromosome / Q000648:ultrastructure