Dab enhancer

hESCs were treated with digoxin (2.5 μM), lanatoside C (2.5 μM), or DMSO control for 24 hours. Approximately 10⁶ treated cells were mixed with 10⁵ MEFs to promote teratoma formation in 50 μl PBS^{56 (link)}. The cells mixture and 1x Matrigel Matrix was mixed well and the cells were subcutaneously injected into NOD scid gamma mice (NSG mice) for 8 weeks. After 8 weeks, animals were sacrificed and teratoma was removed, fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin. H&E stain protocol was modified from previous study^{57 (link)}. For immunohistochemistry, teratoma sections were blocked using 5% milk for 1 hour, and stained with primary antibody at 4 °C overnight, follow by secondary antibody (Dako, Santa Clara, CA, USA) for 1 hour at RT and DAB enhancer (Dako). Primary antibody: anti-human alpha-1-fetoprotein (A0008, Dako) for endoderm lineage; anti-human smooth muscle actin, clone 1A4 (M0851, Dako) for mesoderm lineage; anti-Tuj1 (MAB1637, EMD Millipore, Darmstadt, Germany) for ectoderm.

A proportion of surgical tissue specimens were fixed with formalin paraffin-embedded. IHC staining was performed on 4-μm thick section. Briefly, each slide was incubated with primary antibody against HIF-1α (1:100, Abcam, UK, Cat. #ab51608) or c-myc (1:200, Abcam, UK, Cat. #ab32072) overnight after a series of procedures (de-paraffin, antigen retrieval, rinse). It was followed by an incubation with the anti-rabbit IgG-HRP antibody (1:1000, Sungene Biotech, China, Cat. #LK2001) for 30 minu. The membrane was then washed five times with TBST and enriched with the brown color of DAB Enhancer (Dako). The expression of HIF-1α or c-myc was evaluated by three experienced pathologists. The “positive” result in IHC images showed that the nucleus and cytoplasm of the cells were yellow or brownish yellowes fine particles. The “negative” result showed no evidence of yellow or brownish yellow particles in nucleus and cytoplasm.