Anti notch4

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)].

Cells were lysed using RIPA buffer, and BCA protein assay kit (Beyotime, Shanghai, China) was utilized to calculate the protein concentration. Then cellular proteins were subjected to SDS‐PAGE gels and transferred to polyvinylidene difluoride membrane (Millipore, USA). Blots were blocked with 5% nonfat milk for an hour, before incubating with primary antibodies overnight at 4°C followed with appropriate secondary antibodies for an additional an hour at room temperature. The protein band was detected by ChemiDoc™ XRS + system and Image J software were applied for determination of the protein abundance. Primary antibodies used in Western blot analyses included anti‐GHITM (Proteintech, China, 16,296‐1‐AP), anti‐YY1 (Proteintech, China, 22156‐1‐AP), anti‐PD‐L1 (Proteintech, China, 28076‐1‐AP), anti‐Notch1 (Proteintech, China, 20687‐1‐AP), anti‐Notch2 (Proteintech, China, 28580‐1‐AP), anti‐Notch3 (Proteintech, China, 55114‐1‐AP), anti‐Notch4 (Abcam, UK, ab184742) and anti‐GADPH (Proteintech, China, 10494‐1‐AP).