Metamorph imaging analysis software

Telomere lengths were measured on paraffin-embedded sections of lung tissue by Quantitative Fluorescence in Situ Hybridization (Q-FISH). Briefly, after deparaffinixation, tissues were suspended in 10mM sodium citrate buffer, pH 6.5, heated in a microwave, then incubated for 15 min in 0.01M HCL containing 1% pepsin (Thermofisher Scientific, South San Francisco, CA). The tissues were washed then treated with 10mg/ml RNase solution (Qiagen, Hilden, German). After washing, the tissues were incubated with 0.3 μg/ml PNA probe TelC-Cy3 (Panagene, Daejeon, Korea) suspended in formamide buffer (70% formamide, 10 mmol/L Tris, pH 7.5), heated to 78°C for 10 min then incubated overnight at 20°C. The tissues were then washed sequentially with formamide buffer then PBS containing 0.1% Tween, blocked with 3% BSA (Sigma, St. Louis, MO), 10% donkey serum and incubated overnight at 4°C with rabbit-anti SPC antibody (MilliporeSigma). Tissues were washed with PBS containing 0.1% Tween and incubated secondary antibody at 20°C for 1 h, washed, and mounted using prolong gold anti-fade mounting medium with DAPI (Life Technologies). Images were acquired using a Zeiss Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) and telomere signal intensity was quantified in a blinded manner using MetaMorph imaging analysis software (Molecular Devices, Sunnyvale, CA).

The livers and pancreases of all recipients were examined at necropsy. Additionally, specimens from the 3-4-5 GE donor pancreases prior to islet isolation and from transgenic hCD46 pigs used as donors in our previous study (18 (link)) were examined. Tissue sections were fixed in both 10% buffered formalin and 4% paraformaldehyde. Formalin-paraffin-embedded sections were stained with hematoxylin/eosin, using standard procedures. Paraformaldehyde-fixed tissues were used for immunofluorescence analysis. Primary and secondary antibodies are listed in Table 4. Nuclear staining was done with TO-Pro-3 iodide (Molecular Probes, Eugene, OR).
Images were captured by a Photometrics Cool SNAP digital camera (Roper Scientific, Tucson, AZ) and Nikon C1 confocal system at 40x objective lens (Nikon Instruments, Melville, NY), and analyzed by MetaMorph imaging analysis software (Molecular Devices, Downington, PA). Photographs were taken through a Nikon Eclipse E800 microscope (Nikon Instruments).