Lsm 510 nlo laser scanning microscope

For intravital microscopy (IVM), we adapted the previously mentioned transplantation protocol. Fluorescent-labeled HHALPCs (CellTracker Red CMTPX Dye, C34552, Thermo-Fischer Scientific) at a higher dose of 5 × 10⁷ cells/kg were transplanted by intraportal injection into adult Wistar rats (n = 3/group). Liver vascularization was assessed at different time points (1 h, 24 h, 48 h and 7 days) after transplantation by intravital microscopy (S1 Supplementary Data). Liver vasculature and cell nuclei were stained by intravenous injection of 5 mg of 70kDa FITC-dextran (46945, Sigma, St-Louis, MO, USA) and 0.2 mg Hoechst 33342 (H1399, Invitrogen, Carlsbad, CA, USA). Images were collected using an LSM 510-NLO laser scanning microscope (Zeiss, Oberkochen, Germany) in multiphoton mode, equipped with a 25× LD-objective NA0.8. For fluorescence excitation, a laser line of 800 nm (2%) was used, and detection was through 3 emission channels (Metadetector window 585–628 nm for red emission; two bandpass filters BP 435–485 for blue, and BP 500–550 for green emission). Z-stacks were collected to reconstruct 3D images using Zen 2012 image processing software (Zeiss).

Unmolded PDMS chips were stained with 25 μg/ml 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a fluorescent lipophilic tracer, in phosphate buffer solution (PBS). The PDMS microdevices were covered with the staining solution at room temperature for 20min, and then washed three times for 5min to remove excess staining. Then, culture medium was added and spheroids were deposited in the centre of the microdevices.
Confocal acquisitions were done using Zeiss LSM 510 NLO laser-scanning microscope, fitted with a water immersion 20X objective.