Low Magnification Confocal Microscopy of Tumor Angiogenesis
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Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI. We provide details on how to image fluorescently labeled blood vessels.
Key wordsBlood vessels Angiogenesis Fluorescent lectin DiI Confocal microscope
GM thanks Bob Zucker (US EPA, Research Triangle Park, NC) for low power inspiration and discussions, and Thomas D. Coates (CHLA) for direction. We thank Shinya Yamada (CHLA and Tokai University Hachioji Hospital, Tokyo, Japan) for the latex vascular cast method. We thank Clark Thom and Klaus Schreck (Leica Microsystems, Exton, PA) for confocal on-site service, Kolja Wawrowsky (Cedars-Sinai Medical Center Los Angeles, formerly with Leica) for confocal training, Frank Lie, Scott Young, Rob Dunakin, David Zemo, Bob Vogel, and Chris Kier for confocal technical and application support. We are grateful to Bob Vogel and Martin Hoppe of Leica Microsystems for permission to include LCS Lite software with the Paddock 2.0 book CD-ROM. LCS Lite is available for free download from the Leica Microsystems Web site and from Leica salespeople and dealers. Experiments involving multiphoton excitation of Hoechst 33342 were conducted on a Zeiss LSM 510 META NLO microscope at the Light Microscopy Core of City of Hope National Medical Center (http://www.cityofhope.org/LMC/LSM510.asp), in collaboration with Dr. Christine Brown, Renate Starr and Prof. Michael Jensen.
We are grateful to Sam Gambhir for providing the tribrid hrLuc-DsRed2-TK reporter gene construct prior to publication (Ray et al. ). We thank Denise Petersen, Karen Pepper, and Don Kohn, CHLA Gene Vector Core, for inserting the tribrid reporter gene into the lentivirus vector and transducing U87MG cells. We thank Dr. Ignacio Gonzalez for histology slide preparation and tissue diagnoses. Our thanks to Ignacio Gonzalez, Dr. Rex Moats, Dr. Mike Rosol, Maya Otto-Duessel, and Dr. Shawn Chen, for discussions.
This work was supported by grants from the National Institutes of Health (CA 82989 to W.E. Laug), the T.J. Martell Foundation (New York) (to W.E. Laug), and an U.S. HRSA capital equipment grant (to Y. DeClerck). Confocal and standard fluorescence microscopy was performed in the Congressman Julian Dixon Cellular Imaging Facility of Children’s Hospital Los Angeles. Hoechst 33342 multiphoton imaging at City of Hope National Medical Center was done in collaboration with Dr. Christine Brown, and Renate Starr, and was funded by NIH grants to Professor Michael Jensen.
The University of Miami Leica SP5 and MP-NDD4/SP5/FCS/FLIM confocal microscopes were purchased with funds from the Diabetes Research Institute Foundation. Rong Wen and Yiwen Li are supported by the National Eye Institute and Bascom Palmer Eye Institute.
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