Ab134161

Human NB tissue samples (obtained from Children’s Hospital of Fudan University with full consent after approval from Local Ethics Committee, No. 2017-66) were fixed with 4% formaldehyde. Paraffin-embedded tumor tissues were sectioned to 5 µm thickness and mounted on positively charged microscope slides. And 1 mM ethylene diamine tetraacetic acid (pH 8.0) for B7H3 was used for antigen retrieval. Endogenous peroxidase activity was quenched by incubating the slides in methanol containing 3% hydrogen peroxide, followed by washing in phosphate buffered saline (PBS) for 15 minutes. The sections were incubated for 30 minutes at 37℃ with normal goat serum and subsequently incubated at 4℃ overnight with primary B7H3 antibodies (1 : 200, ab134161; Abcam, Cambridge, MA, USA). The sections were then rinsed with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse antibodies, followed by reaction with diaminobenzidine and counterstaining with Mayer's hematoxylin. The Immunoreactive Score (IRS) was used for B7H3 IHC evaluation and score from 0 to 12 points was obtained by two separate pathologists. Samples with IRS values between 0 to 2 were considered negative, 3 to 5 were positive (+), 6 to 8 were moderate positive (++), 9 to 12 were strong positive (+++).

Stable cells were washed twice with PBS and suspended in a lysis buffer (2% Mercaptoethanol, 20% Glycerol, 4% SDS in 100 mM Tris-HCl buffer, pH 6.8). Equal amount of proteins were loaded and separated by SDS-PAGE, and then transferred onto PVDF membrane (Schleicher&Schuell Co, Keene, NH, USA) using an electro- blotting apparatus (Bio-RAD, Hercules, CA, USA). The membrane was blocked with 5% nonfat milk (R&D, Minneapolis, MN, USA) in TBST solution for 1 hour at room temperature, and incubated overnight at 4℃ with specific antibody to B7H3 (1 : 1000, ab134161; Abcam), cyclinD1 (1 : 1000, ab134175; Abcam), Rb (1 : 1000, ab181616; Abcam), E2F1 (1 : 1000, ab179445; Abcam). After three washes in TBST solution, the membrane was incubated with secondary antibody diluted with TBST solution at room temperature for 2 hours. The signals of detected proteins were visualized on Image Quant LAS 4000 mini detection system (GE, Boston, MA, USA). β-actin were used as a loading control.