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Protocol to assess growth plate kinetics during long‐term in vivo multiphoton imaging using BrdU as a physiological marker
Author(s) -
Liu Wenhua,
Williams Rebecca M.,
Farnum Cornelia E.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.649.5
Subject(s) - in vivo , biomedical engineering , chemistry , kinetics , fluorescence lifetime imaging microscopy , nuclear medicine , medicine , fluorescence , biology , physics , microbiology and biotechnology , quantum mechanics
With the ability to collect optical images deep into biological tissues, multiphoton microscopy (MPM) is a powerful tool to study growth plate kinetics in real time, but the act of imaging introduces variables with the potential to alter chondrocytic kinetics. This study is to develop a procedure to assess chondrocytic viability during in vivo MPM imaging on the proximal tibial growth plate of 4‐wk‐old mice. The BrdU labeling index is used as a physiological marker to assess imaging variables, following IACUC procedures. Isoflurane anesthesia was used at 1L/min (no ventilation) to maintain breathing at ~1.5/sec. Fluorescein was injected IP for a vascular concentration of ~1mM. After 2 hrs, a second dye injection was given with ~200 ul of isotonic fluids to avoid hydropenia. Body temperature was maintained at ~35 0 C. The surgical limb was bathed in warm Ringer's. BrdU was given IP 0.5 hr before euthanasia. Fixation and plastic embedment followed, with analysis of BrdU LI by standard protocols. Maintenance of core and limb temperature, hydration, reinjection of the dye after 2 hrs, and limiting imaging to 3.5 hrs proved to be key to successful long‐term imaging. This standard protocol for BrdU‐based analysis of proliferative capacity will allow comparative assessment of multiple vital dyes for monitoring chondrocytic dynamics during multi‐hour MPM imaging. Supported by NIH‐R01AR052003. Grant Funding Source NIH