Ki 67 8d5

Tumor tissues were obtained from tumor-bearing mice treated with one dose of BETd-260 5 mg/kg or vehicle control. The following antibodies were used for IHC: BRD2 (A302-583A, 1:250), BRD3 (A302-368A, 1:250), BRD4 (A700-005, 1:100) from Bethyl Laboratories (Shanghai, China); cleaved PARP-1 (Asp214) (5625, 1:100) and Ki67 (8D5) (9449, 1:500) from Cell Signaling Technology (CST, Shanghai, China). IHC was performed following a standard protocol. Briefly, the 5-µm sections were de-paraffinized with xylene, rehydrated in graded concentrations of ethanol, and boiled in antigen retrieval buffer (Abcam, Shanghai, China) in a microwave oven for 5 min. Slides were incubated with specific primary antibodies at room temperature for 2 h. After incubation, the slides were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated antibody (Invitrogen, Shanghai, USA) at room temperature for 30 min, followed by incubation with ABC (avidin-biotin complex, Vectorlabs, Shanghai, China) for 30 min and visualization by the addition of 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Dako Diagnostics Co., Ltd., Shanghai). Sectioned tissues were counterstained with hematoxylin, dehydrated through a graded series of alcohol into xylene, and mounted under glass coverslips. Images of stained slides were captured using a standard light microscope.

Following BME-2 dissociation, organoids were gently pelleted and fixed in 10% neutral formalin before paraffin embedding and sectioning. Paraffin embedded sections of 3.5 μm were stained by a Bond Max autostainer according to the manufacturer’s instruction (Leica Microsystems). Primary antibodies cytokeratin (AE1/AE3, 1:100, Dako), Ki67 (8D5, 1:400, Cell Signalling Technology), and p53 (D07, 1:50, Leica) were applied with negative controls as previously described^{2 (link)}.

IHC was performed as we described previously (7 (link)). Xenograft tumor tissues were obtained from Balb/c tumor-bearing mice treated with single dose of 5 mg/kg BETd-260 or vehicle control. The following antibodies were used for IHC: BRD2 (IHC-00612), BRD4 (HC-00396), BAD (A302-384A) from Bethyl Laboratories (Shanghai, China); BRD3 (ab264420) from Abcam (Shanghai, China); cleaved PARP (Asp214) (#32563), activated caspase-3 (#9664), and Ki-67 (8D5) (9449) from Cell Signaling Technology (CST, Shanghai, China), Anti-Mcl-1 (MAB828) from R&D Systems (R&D Systems, Shanghai, China).