Rabbit monoclonal anti notch1 antibody

An ElivisionTM super HRP IHC kit (cat. no. Kit‐9921; Fuzhou Maxim Biotechnology Development Co., Ltd.) was used for immunohistochemistry. The paraffin‐embedded sections were deparaffinized, rehydrated and antigen retrieval was performed by heat mediation in 0.01 M sodium citrate, pH 6.0. The sections were incubated overnight with mouse polyclonal anti‐STAT3 antibody (1:500; Cell Signaling Technology, Inc.), rabbit polyclonal anti‐Jagged1 antibody (1:200; Abcam, Inc.), rabbit monoclonal anti‐Notch1 antibody (1:150; Cell Signaling Technology, Inc.), or rabbit monoclonal anti‐Notch2 antibody (1:100; Cell Signaling Technology, Inc.) at 4°C following the instructions on the product datasheets for the primary antibodies. Following incubation with streptavidin peroxidase (Maxim Biotechnology Development Co., Ltd.) for 10 minutes at room temperature, secondary antibodies were added for 30 minutes at 37°C. The immune reaction was then visualized using 3,3'‐diaminobenzidine (DAB) (Fuzhou Maxim Biotechnology Development Co., Ltd., Fuzhou, China). The expression intensity was evaluated by a semi‐quantitative system to calculate the percentage of positive neoplastic cells: 0 points, no positive cells; 1 point, 1% to 25%; 2 points, 26% to 50%; 3 points, 50% to 75%; 4 points, >75%. Then, ≤2 points were judged as negative for expression, 2 points, were judged as positive expression.

Whole cell lysates were prepared from frozen xenograft samples or cell lines using Mammalian Protein Extraction Reagent (Thermo Scientific) lysis buffer supplemented with inhibitors of endogenous protease, kinase, and phosphatase activity (all obtained from Sigma-Aldrich). Twenty micrograms of protein from each sample were separated on 7.5 or 10% polyacrylamide gels and transferred to a PVDF membrane. Following transfer, membranes were blocked with 5% milk in 1× TBS, 0.1% Tween-20 (TBST) and incubated in diluted (1:1000) primary antibody overnight, according to the manufacturer’s recommendations. Primary antibodies used were a rabbit monoclonal anti-Notch1 antibody, a rabbit monoclonal anti-cleaved Notch1 (Val1744) antibody, and a rabbit monoclonal anti-Notch3 antibody (Cell Signaling). Membranes were then incubated with a horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) and developed using a chemiluminescent detection reagent obtained from GE Healthcare Life Sciences. Equivalent protein loading was verified by stripping the blots and re-probing with a mouse anti-Pan-Actin antibody (NeoMarkers).

The protein levels of Notch1 and cleaved Notch1/Notch1 intracellular domain (NICD1) were detected using western immunoblotting. Total proteins were extracted from mouse liver tissues in a protein lysis buffer containing 20 mM Tris at pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 2.1 μM leupeptin, 1 mM PMSF and 1% (v/v) Triton X-100. Extracted proteins were quantified and separated in a 10% SDS polyacrylamide gel as previously described [23 (link),24 (link)]. Following electrophoresis and electrotransfer onto nitrocellulose membrane, the blots were probed with rabbit anti-Notch1 monoclonal antibody or rabbit anti-NICD1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA). The membranes were then reprobed with rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology) to ensure equal loading of the samples. All the blots were incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (Cell Signaling Technology). Proteins were visualized by using an ECL detection system (Bio-Rad, Hercules, CA, USA) and quantified using Quantity One software version 4.6.8 for Windows (Bio-Rad).