Anti dll1

Cells were lysed in cell lysate, and then centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was collected and denatured. Proteins were separated in 10% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (PVDF). The PVDF membrane was treated with TBST containing 50 g/L skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies: anti-CTR2 (1:200, Novusbio), anti-BCL-2 (1:500, Immunoway), anti-OCT-4 (1:1000, Proteintech), anti-KLF4 (1:500, Proteintech), anti-MDR1 (1:200, Santa),, anti-Notch1 ICD (1:1000, Abcam), anti-Notch2 ICD (1:1000, Abcam), anti-Jagged1 (1:500, Abcam), anti-Jagged2 (1:1000, Abcam), anti-DLL1 (1:500, Abcam), anti-DLL4 (1:500, Abcam), anti-Notch ICD (1:1000, Cell Signaling), anti-SDF-1 (1:1000, Abcam), anti-CXCR4 (1:2000, Abcam) and anti-β-actin (1:1000, Cell signaling) respectively, at 37 °C for 1 h. Membranes were rinsed and incubated for 1 h with the correspondent peroxidase-conjugated secondary antibodies. Chemiluminent detection was performed with the ECL kit (Pierce Chemical, Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of ß-actin.

Immunohistochemistry was conducted by Tissue microarray analysis (TMA) of 76 specimens as previously described (20 (link)) following evaluation of tumor content and quality in hematoxylin and eosin-stained sections. TMAs were processed in a Leica BOND-III (Leica Biosystems, Nussloch, Germany) automated, continuous random, access slide-staining system that simultaneously processes multiple immunohistochemistry (IHC) assays. Protocol F was selected, with 3 min of heat-induced epitope retrieval and Bond Polymer Refine Detection (Leica Biosystems, DS9800) of primary antibodies. Anti-Notch1, anti-Notch2, anti-Notch3, anti-Notch4, anti-DLL1, anti-DLL2, anti-DLL3, and anti-SSTR2/5 (Abcam, Cambridge, UK) were the primary antibodies. Expression was assayed in photographs taken with an Aperio AT2 whole slide scanning system (Leica Biosystems). Staining intensity was scored as 0, no staining; 1, weak; 2, moderate; and 3, strong staining. An H-score was calculated from the percentage of positively stained cells at each intensity level using the following formula: [1 × (% weakly stained cells) + 2 × (% moderately stained) + 3 × (% strongly stained cells)].