Mandible, radius, ulna, vertebra (3rd lumbar, L3), distal
femur, and proximal tibia were assessed for whole bone fluorescence using
reflectance epi-fluorescence imaging (IVIS SpectralCT, PerkinElmer). Our lab and
others have previously used this method as an assay to measure
fluorescently-tagged bisphosphonate levels in tissues [17 (link),25 (link),27 (link),28 (link)]. Mandible, radius, and ulna were scanned whole whereas,
vertebrae processes were removed, and distal femur and proximal tibiae were cut
to 10 mm standard length sections. For each skeletal site, a bone from each
animal was run on a single plate to assume uniform scan setting. While this
allows comparison across animals within bone site, the optimization of settings
to accommodate for the size differences among bone sites restricts the ability
to compare levels across bone sites. Exposure time was approximately one second
per sample plate using broad emission spectral excitation and emission ranging
of 430–465nm and 500–540nm, respectively. FAM-ZOL signal was
distinguished from auto fluorescence and calcein blue signals by spectrally
unmixing the image series (Living Image, PerkinElmer). In all cases, bone
fluorescence levels are reported as average radiant efficiency
([photons/second]/[μW/cm2]).
femur, and proximal tibia were assessed for whole bone fluorescence using
reflectance epi-fluorescence imaging (IVIS SpectralCT, PerkinElmer). Our lab and
others have previously used this method as an assay to measure
fluorescently-tagged bisphosphonate levels in tissues [17 (link),25 (link),27 (link),28 (link)]. Mandible, radius, and ulna were scanned whole whereas,
vertebrae processes were removed, and distal femur and proximal tibiae were cut
to 10 mm standard length sections. For each skeletal site, a bone from each
animal was run on a single plate to assume uniform scan setting. While this
allows comparison across animals within bone site, the optimization of settings
to accommodate for the size differences among bone sites restricts the ability
to compare levels across bone sites. Exposure time was approximately one second
per sample plate using broad emission spectral excitation and emission ranging
of 430–465nm and 500–540nm, respectively. FAM-ZOL signal was
distinguished from auto fluorescence and calcein blue signals by spectrally
unmixing the image series (Living Image, PerkinElmer). In all cases, bone
fluorescence levels are reported as average radiant efficiency
([photons/second]/[μW/cm2]).