Paraplast x tra paraffin

1×10⁶ of WT or Klf5-overexpressing ESCs were injected into the dorsal flanks of 6–7-week-old immune-deficient NCr-nu/nu female mice (Taconic, Cat# NCRNU). After 4–5 weeks, resulting teratomas were collected by surgical removal, fixed overnight in 10% buffered formalin (Fisher Scientific, Cat. # SF100–4), dehydrated in a graded series of ethanol solutions, embedded in Paraplast X-TRA paraffin (Fisher Scientific, Cat. # 23–021-401), sectioned at 6 mm thickness, and stained with hematoxylin and eosin (H&E) using standard procedures (Choi et al., 2011 (link)). These paraffin sections will be subjected to immunohistochemistry

All samples were collected and fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂ (pH 7.4) for 4 h at room temperature and then transferred to 0.1 M sodium cacodylate buffer with 10 mM CaCl₂ (pH 7.4) for 24 h at room temperature to re-crosslink alginate. Then samples were dehydrated through a series of ethanol washes followed by two Citrisolv (Fisher Scientific, Pittsburgh, PA) washes. The samples were embedded in Paraplast X-tra Paraffin (Fisher Scientific) and sectioned to 5mm thick sections and placed on positively charged glass slides (Fisher Scientific). Prior to staining, sections were oven-dried at 37°C for 2 h, deparaffinized in Citrisolv, and rehydrated in decreasing ethanol washes. Samples were stained using alcian blue (Poly Scientific, Bay Shore, NY) for 30 min, followed by standard washes. Samples were counterstained under nuclear fast red (Poly Scientific) for 5 min. After dehydration and clearance, the slides were mounted and imaged.