Ab103590

Cells grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. After blocking with BSA, cells were incubated with primary antibody at 4 °C overnight followed by incubation with fluorescent secondary antibody for 2 h at room temperature. 4’,6-diamidino-2-phenylindole (DAPI) (0.1%) was used to label the nucleus for 15 min. Cells were examined under a confocal laser microscope (Leica TCS SP5II STED, Mannheim, Germany). At least 200 cells on each slide were counted, and the percentage of cells with targeting protein was calculated. The primary antibodies used are shown below: ANGPTL4 (Abcam, ab196746), ESM1 (Abcam, ab103590) and CD34 (Abcam, ab81289).

For immunofluorescence staining of ESM1 and ANGPT2, paraffin-embedded 3 μm serial sections of five cases of laryngeal or hypopharyngeal squamous cell carcinoma samples were deparaffinized and rehydrated. Preheat EDTA 8.0 was used for repairing in the high pressure cooker. Polyclonal rabbit anti-human primary antibodies anti-ESM1/FITC (ab103590, Abcam, Cambridge, England) and anti-ANGPT2/TRITC (Abcam, Cambridge, England) (1:100) were applied overnight at 4 °C. After washing, fluorescently conjugated secondary antibodies were used. Nuclear counterstain was achieved using DAPI staining. All fluorescently stained images were taken using an Olympus BX-51 upright light microscope (Olympus, Tokyo, Japan). Each site was imaged in all channels and overlaid in DPViewer version before examination in Photoshop.

Cells were harvested and lysed in a RIPA buffer, and centrifuged at 16,000× g for 20 min at 4 °C. The supernatants were collected to determine protein concentration using the Bradford method. Equal amounts of protein were subjected to 8–10% SDS-PAGE for 2 h at 100 V. The separated proteins were transferred onto Hybond-P+ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with the following primary antibodies: anti-cyclin D1 (ab16663; 1:1000, Abcam), anti-cyclin B1 (ab32053; 1:1000, Abcam), anti-cleaved caspase-3 (9661S; 1:1000, Cell Signaling Technology), anti-pH2AX (2577S; 1:1000, Cell Signaling Technology), anti-CD44 (ab51037; 1:1000, Abcam), anti-ESM1 (ab103590; 1:1000, Abcam), and anti-OCT3/4 (sc-9081; 1:1000, Santa Cruz Biotechnology) antibodies. The bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and an ECL (Bio-Rad) Western blotting detection reagent. The relative protein levels were normalized to those of β-actin (MA5–15739, Thermo Fisher Scientific, Waltham, MA, USA) used as a loading control.

Antibody against ESM1 (ab103590) was purchased from Abcam (Cambridge. MA). Antibodies against p21 (sc-397; 1:1000), MMP-2 (sc-53630; 1:1000), MMP-9 (sc-6840; 1:1000), uPA (sc-14019; 1:1000), cyclin D1 (sc717; 1:1000), and β-actin (sc-47778; 1:2000) were purchased from Santa Cruz Biotechnology (Dallas, Texas). Horseradish peroxidase conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Promega (Madison, WI). MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] was purchased from Sigma (St. Louis, MO). Recombinant Human TIMP-1 Protein was purchased from R&D Systems, Inc (Minneapolis, MN). Antibody against TIMP-1(#8946; 1:1000) was purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).