Horseradish peroxidase hrp conjugated antibody

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.

A tissue microarray of 40 OS tissue sections and normal bone tissue sections was purchased from Alenabio Biotechnology (Xi'an, China). Tumor tissues were obtained from tumor-bearing mice treated with MLN4924 or vehicle control for 3 days. The antibodies used for IHC, p27 (2552), and p21 (2947) were purchased from Cell Signaling Technology (CST, Shanghai, China). IHC was performed following a standard protocol. Briefly, the sections were de-paraffinized by 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 diluted antibodies at room temperature for 2 h. After incubation, the slides were washed three times with PBS, 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 (Shanghai) Co., Ltd.), with hematoxylin as the counter stain. Images of stained slides were captured using a standard light microscope.

The cells were lysed with a protein lysis buffer (Pro-PREP, Intron Biotechnology, Korea) with a protease/phosphatase inhibitor cocktail, and protein concentration were determined using a DC assay kit (Bio-Rad, Berkeley, USA). The proteins were separated via 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, blocked with 3% bovine serum albumin in Tris-buffered saline with Tween (TBST: 20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 1% Tween 20), and probed with the indicated primary antibodies; rabbit anti-p16^INK4a (1:1000; Abcam, Cambridge, UK; ab108349), rabbit anti-BMI1 (1:1000, Cell Signaling Technology Europe, Leiden, The Netherlands; #6964s), mouse anti-GAPDH (1:3000, Millipore, Darmstadt, Germany; MAB374), rabbit anti-COX-2 (1:1000, Abcam; ab15191), rabbit anti-p-p38 (1:1000, Cell Signaling; #9211s), rabbit anti-MKP-1 (1:200, Santacruz, Texas, USA; sc-1102). The secondary antibodies were used according to the manufacturer's specifications; horseradish peroxidase (HRP)-conjugated antibodies (1:2000; Invitrogen, Carlsbad, USA; G21040, G21234) and binding was detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotek, Amersham, UK).

The protein samples were prepared with the protein lysis buffer Pro-pro (Intron Biotechnology, Korea), and the mitochondrial fraction was isolated using a mitochondria isolation kit (Thermo Scientific, USA) according to the manufacturer’s specifications. The protein concentration was determined using bovine serum albumin (BSA) as the standard (Bio-Rad Laboratories, Hercules, USA). The proteins were separated via 10 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with 3% bovine serum albumin in Tris-buffered saline with Tween (TBST: 20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 1% Tween 20), the blots were probed overnight at 4 °C with the primary antibodies. The secondary antibodies, horseradish peroxidase (HRP)-conjugated antibodies (1:2000; Invitrogen, USA), were incubated with the membranes at room temperature for 1 h and detected using enhanced chemiluminescence (ECL) detection kit (GE Healthcare Life Science, UK). The primary antibodies used are listed in Supplementary Table 1.

Breast tissue samples were obtained at the time of surgery. Immunohistochemistry (IHC) analyses were performed on 4 μm, formalin-fixed, paraffin-embedded slides from breast cancer tissues and matched non-cancerous tissues. Paraffin-embedded tissue sections were deparaffinized, rehydrated, rinsed, and immersed in 10 mM sodium citrate (pH 6.0) for antigen retrieval under high pressure in a pressure cooker for 3 minutes. After treated with in methanol containing 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, the slides were incubated with primary antibody for 1 hour at 37°C. After washing, sequential incubations were performed with horseradish peroxidase (HRP) conjugated antibodies (Invitrogen) for 30 min at room temperature. The stain was visualized using DAB Plus (Dako) and hematoxylin counterstain. The expressions of S100A16, ER, PR, HER2 were all detected by IHC.
A Fluorescence in situ hybridization (FISH) test for HER2 gene amplification was routinely ordered when HER2 was IHC 2+. FISH was performed using the PathVysion HER2 DNA FISH Kit (Vysis Inc, Downers Grove, IL), according to the manufacturer’s instructions.