All mice were housed according to IACUC guidelines and used for experiment when 8–14-weeks old. Wild-type C57BL/6.SJL mice were HSPC donors when recipients were wild type, Col2.3–GFP or WWv double mutant (backcrossed to C57BL/6 background). FVB mice were donors for wild-type or PPR littermate mice4 (link),29 (link) (gift from E. Schipani).
Mice were anaesthetized and prepared for in vivo imaging as described3 (link). Immediately before imaging 20 µl of non-targeted Qdot 800 or 655 (Invitrogen) diluted in 130 µl sterile PBS was injected retro-orbitally to allow vasculature visualization. The mouse was held in a heated tube mounted on a precision 3 axis motorized stage (Suter MP385). All mice were imaged with a custom-built confocal two-photon hybrid microscope specifically designed for live animal imaging (see Methods). At the start of each imaging session, we surveyed large areas of the skull bone surface using video rate second harmonic microscopy (see Methods) to identify the major anatomical landmarks such as sagittal and coronal sutures. We identified the locations of HSPCs within bone-marrow cavities and recorded their coordinates relative to the intersection of the sagittal and coronal sutures. SHG and GFP signals above each identified HSPC were acquired every 5 to 20 µm until the above endosteal surface was reached. After in vivo imaging, the scalp was re-closed using 3 M Vetbond veterinary glue and post-operative care was provided as described3 (link).
Images were coloured and merged using Adobe Photoshop and HSPC-microenvironment distance measures were obtained using Adobe Illustrator and Microsoft Excel. A two-tailed type 2 t-test was applied to all data. P values ≤0.05 were considered statistically significant.
Mice were anaesthetized and prepared for in vivo imaging as described3 (link). Immediately before imaging 20 µl of non-targeted Qdot 800 or 655 (Invitrogen) diluted in 130 µl sterile PBS was injected retro-orbitally to allow vasculature visualization. The mouse was held in a heated tube mounted on a precision 3 axis motorized stage (Suter MP385). All mice were imaged with a custom-built confocal two-photon hybrid microscope specifically designed for live animal imaging (see Methods). At the start of each imaging session, we surveyed large areas of the skull bone surface using video rate second harmonic microscopy (see Methods) to identify the major anatomical landmarks such as sagittal and coronal sutures. We identified the locations of HSPCs within bone-marrow cavities and recorded their coordinates relative to the intersection of the sagittal and coronal sutures. SHG and GFP signals above each identified HSPC were acquired every 5 to 20 µm until the above endosteal surface was reached. After in vivo imaging, the scalp was re-closed using 3 M Vetbond veterinary glue and post-operative care was provided as described3 (link).
Images were coloured and merged using Adobe Photoshop and HSPC-microenvironment distance measures were obtained using Adobe Illustrator and Microsoft Excel. A two-tailed type 2 t-test was applied to all data. P values ≤0.05 were considered statistically significant.
