Mouse linker

hUCESCs were cultured as described above. 3 × 10⁴ cells were seeded in slides, and fixed for 10 minutes in 96% ethanol, before processing for immunocytochemistry. Mouse tumors were immersion-fixed in 10% neutral buffered formalin for 24 hours and routinely embedded in paraffin. 4 μm thick sections were mounted on Flex IHC microscope slides (Dako, Glostrup, Denmark). The immunohistochemical (IHC) techniques were automatically performed in an AutostainerLink 48 (Dako). FLEX ready-to-use Dako primary antibodies to CK (clone AE1/AE3), E-cadherin (clone NCH-38), vimentin (clone V9), desmin (clone D33), actin (clone HHF35), smooth muscle actin (clone 1A4), β-catenin (clone beta-catenin-1) were employed. KLF4, OCT4, and Sox2 primary antibodies were obtained from Santa Cruz Biotechnology (Dallas, USA), Millipore, and Sigma-Aldrich, respectively. Epitope retrieval was performed in a PT Link (for 20 minutes at 97ºC) using EnVision FLEX target retrieval solution (pH 9). All antibodies were incubated for 20 minutes at RT. As detection system we used EnVision FLEX/HRP Dako (dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat anti-mouse and anti-rabbit immunoglobulins) for 20 minutes. For E-cadherin, a mouse linker (Dako) was added. Quantitation of immunopositive cells for active caspase-3 expression was performed as previously described [57 (link)].

Paraffin-embedded, formalin-fixed, 3-μm tissue sections from human HPC patients were deparaffinized in xylene, and rehydrated by washing with a series of ethanol dilutions and distilled water. Heat-induced epitope retrieval was performed by microwaving the sections in a pH 9.0 target retrieval solution (Dako, Denmark) for CD271 staining or 10 mM citric acid buffer (LSI Medience Corporation, Japan) for involucrin staining. Endogenous peroxidases were blocked with 0.3% H₂O₂. The sections were incubated with anti-human CD271 (1:4,000) for 20 min, or with anti-involucrin (1:1,000) for 90 min, at 37 °C. The CD271-stained sections were incubated for 15 min with mouse LINKER (Dako), followed by incubation with secondary antibody, and development with 3,3′-diaminobenzidine (DAB) Chromogen (EnVision™ Detection SystemsPeroxidase/DAB, Rabbit/Mouse, Dako). Immunostaining with anti-CD271 and anti-involucrin was performed according to the manufacturers’ protocols. Anti-Ki67 staining was performed on a Ventana Discovery automation system (Roche, Switzerland).

For IGFBP2 immunostaining, paraffin-embedded, formalin-fixed 3-μm tissue sections from human RCC patients were deparaffinized in xylene and rehydrated by washing with a series of ethanol dilutions and distilled water. Heat-induced epitope retrieval was performed by microwaving the sections in a pH-9.0 target-retrieval solution (Dako, Produktionsvej, Denmark). Endogenous peroxidases were blocked with 0.3% H₂O₂. The sections were incubated with anti-human IGFBP2 for 12 h (1:1,000) at 37 °C, then with mouse LINKER (Dako) for 15 min, followed by incubation with the secondary antibody and development with 3,3′-diaminobenzidine (DAB) chromogen (EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse, Dako).