Lsm 880 indimo

Additional samples from each group were labeled with calcein and calcein blue to evaluate new calcium deposition. Prior to MSC seeding, decellularized bone plugs were soaked in 30 μM calcein (Sigma, St. Louis, MO) for 48 hours to label any existing calcium and washed in PBS for an additional 48 hours to eliminate any unbound calcein. Constructs were generated with calcein-labeled bone plugs, cultured for four weeks, fixed, and stored in 70% ethanol for 48 hours. Samples were removed from ethanol and placed into 30 μM calcein blue (Sigma, St. Louis, MO) for 48 hours to label any new calcium deposition that occurred during culture and washed in 70% ethanol for 48 hours. Simultaneous second harmonic generation (SHG) microscopy and two-photon excited fluorescence (TFEP) of calcein labels was performed based on procedures described previously.^{[43 (link)]} Images were obtained on Zeiss LSM 880 Indimo with Zeiss Axio Observer Z1 inverted stand using a 40×/1.2 C-Apochromat water immersion objective with SpectraPhysics Insight laser at 760nm and non-descanned detectors used for calcein and calcein blue detection. Additionally, acellular, calcein labeled bone plugs were imaged at 0 and 4 weeks to confirm no photobleaching was occurring during the culture period (Figure S3, S4).