In situ imaging of the mouse thymus using 2-photon microscopy.
Two-photon microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37 degrees C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.
Ladi
Ena
E
Department of Molecular and Cell Biology, University of California, Berkeley, USA.
Herzmark
Paul
P
Robey
Ellen
E
D000818:Animals; D000831:Animals, Newborn; D002678:Chimera; D051379:Mice; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D013950:Thymus Gland / Q000648:ultrastructure*