Anti pancytokeratin clone ae1 ae3

To assess the growth of xenograft tumors in nude mice, HSC-3 shRNA Control and HSC-3 shRNA FSCN were injected subcutaneously in the flank or were implanted into the tongue (orthotopic model) of 12 week-old BALB/c nude mice (20 animals/cell line). Animals were sacrificed 3 weeks later and the tumors in the dorsum were dissected, weighted and fixed in 10% formalin for hematoxylin and eosin (HE) stain and immunohistochemistry. Animals, which received the injection of the tumor cells in the tongue, had the tongue and cervical lymph nodes dissected, fixed and subjected to HE stain and immunohistochemistry. Primary tumors (both in flank and tongue) were subjected to immunohistochemical analysis using the anti-fascin antibody, whereas cervical lymph nodes were analyzed with anti-pancytokeratin (clone AE1/AE3; Dako, USA).

Patient tumor material or xenografts were fixed in 4% formalin prior to paraffin embedding. Sections of 5 μm were prepared on a microtome. Tissue sections were deparaffinized and heat mediated antigen retrieval was performed using Tris-EDTA buffer solution pH 9 for Hedgehog staining or 10 mM sodium citrate solution pH 6 for other stainings. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS. Aspecific staining was blocked using Ultra-V Block (Immunologic) for 10 min at room temperature. Primary antibodies were diluted in normal antibody diluent (KliniPath), applied on tissue sections and incubated overnight at 4°C in a humidified chamber. For amplification of signal Brightvision + post antibody block (Immunologic) was used prior to the addition of the secondary antibody; poly-HRP-anti Ms/Rt/Rb IgG (Immunologic) both for 30 min at room temperature. Visualization was performed using Vector® NovaRED™ (Vector Labs) according to manufacturer’s protocol, counterstained with 30% haematoxylin and tissue sections were mounted with non-aqueous medium. Antibodies used for immunohistochemistry were: anti-alpha smooth muscle actin (Abcam, 1:1000); anti-Hedgehog (clone H160, Santa Cruz, 1:1500), anti-Cytokeratin 19 (clone RCK108, BioGenex, 1:1000), anti-pan cytokeratin (clone AE1/AE3, Dako, 1:100).