Cardiogreen

Immunofluorescence staining for confocal microscopy was essentially performed as described before^{61 (link)}. Briefly, iPSCs were seeded on Matrigel-coated coverslips for differentiation and fixed with 4% paraformaldehyde. After permeabilisation with 0.1% Triton X-100 for 5 minutes, cells were incubated in blocking solution (5% BSA, 1% fish skin gelatine, 50 mM Tris in PBS) followed by staining with primary (diluted in blocking solution, 1 h at RT or o/n at 4 °C) and secondary (1 h at RT) antibodies and embedding in Mowiol. Images were acquired using a Zeiss LSM510 confocal microscope equipped with a Plan-Neofluar 40×/1.3 oil objective. For Periodic acid Schiff (PAS) staining, we used the PAS staining kit (Carl Roth) and indocyanine green uptake was analysed by treating living cells with 100 µg/ml Cardiogreen (Sigma) for 30 minutes at 37 °C. Bright-field images were acquired with a Leica DM IL LED microscope.

Mice were anesthetized with 2% isoflurane and injected intravenously with either $60\mu \mathrm{M}$ ICG (Cardiogreen, Sigma-Aldrich, Missouri) or Cypate-cyclo(D-Cys-Gly-Arg–Asp-Ser-Pro-Cys)-Lys-OH (LS301) in $100\text{-}\mu \mathrm{L}$ PBS or Alexa Fluor™ 680 Conjugate (ThermoFisher, Massachusetts). 3-D tumor images were obtained using the FMT 4000 system (PerkinElmer, Inc., Massachusetts) with $1.5\mathrm{mm}\times 1.5\mathrm{mm}$ source density. Reconstruction and image analyses were performed using TrueQuant™ software (PerkinElmer, Inc., Massachusetts). Fluorescence quantification of fluorophores was based on concentration-dependent calibration using the calibration phantom provided with the system. Rectangular prism ROIs were drawn manually around tumors of 3-D reconstructed images, using the topographic projections, stereotactic coordinates, and bioluminescence images for guidance. Quantification of data is given as mean fluorophore concentration in regions of interest, omitting voxels with arbitrary low signal ( $<{10}^{-9}\mathrm{nM}$ ). Background subtracted quantification was obtained by subtracting the mean concentration in the preinjection data from postinjection data.

Indocyanine green (ICG, Cardiogreen, Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) and then added to the medium for 1 h. The final concentration of the resulting ICG solution was 1 mg/mL. After incubation, the medium was exchanged and images representing ICG uptake were taken. After 6 h, the functional ability of hepatocytes to remove the dye was inspected.