Immunohistochemical analysis was carried out using 4 μm paraffin-embedded sections of mouse colon. A standard avidin–biotin peroxidase complex method was employed. Briefly, deparaffinized sections on glass slides were heated in a microwave oven for antigen retrieval, endogenous peroxidase was quenched using 3% H2O2, and tissue sections were incubated at 4°C overnight with primary antibodies. Biotinylated secondary antibody and Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) were applied with 3, 3′-diaminobenzidine. Sections were then counterstained with hematoxylin. Primary antibodies used in the study included: anti-8-oxo-deoxyguanosine (8-oxo-dG) (mouse monoclonal; JalCA), anti-nitrotyrosine (rabbit polyclonal; Millipore), anti-Nuclear Factor Kappa B p65 subunit (NF-κB p65) (rabbit polyclonal, Abcam), anti-phospho-STAT3 (p-STAT3) (rabbit monoclonal, Cell Signaling Technology), anti-phospho-Histone H2AX (γH2AX) (rabbit monoclonal, Cell Signaling Technology), and anti-cleaved caspase-3 (rabbit polyclonal, Cell Signaling Technology). The number of positively stained cells and total number of cells were quantified using the Aperio ScanScope GL system (Vista, CA). About 10,000–30,000 cells from colon tumors or adjacent tissues were analyzed per mouse.
Anti phospho stat3 p stat3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-STAT3 (p-STAT3) is a laboratory reagent used to detect the phosphorylated form of the Signal Transducer and Activator of Transcription 3 (STAT3) protein. It is a specific antibody that recognizes the phosphorylated tyrosine residue on the STAT3 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
Anti nitrotyrosine, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Ruxolitinib, by Cayman Chemical (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Protein lysis buffer, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti survivin, by Abcam (1 mentions)
Anti interleukin 6 il 6, by Abcam (1 mentions)
Anti bcl xl, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
4 protocols using anti phospho stat3 p stat3
1
Immunohistochemical Analysis of Colon Tissue
2
Mesenchymal Stem Cells' Response to Cancer Microenvironment
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BM-MSCs were cultured until to 80–90% confluence and serum-starved overnight using DMEM/LG supplemented with 2 mM L-glutamine and 100 U/mL P/S. Then, the BM-MSCs were treated with MDA CM, MCF7 CM, or CON CM for the indicated times. For JAK and ROS inhibition, the cells were pretreated with ruxolitinib (Cayman Chemical, Ann Arbor, MI, USA) and N-acetylcysteine (NAC; Sigma-Aldrich), respectively, for 30 min prior to the CM treatment. BM-MSCs were rinsed twice with ice-cold PBS and lysed using 2× SDS buffer (100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 2% (v/v) sodium dodecyl sulfate (SDS), 0.001% (w/v) bromophenol blue, and 10% (v/v) β-mercaptoethanol) at 25 °C for 5 min. The cell lysates were collected by scrapping and denatured by heating at 95 °C for 5 min. Western blot analysis was performed with the following primary antibodies: anti-HIF-1α (1:800-1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Stat3 (pStat3; 1:500–1:800; Cell Signaling Technology), anti-Stat3 (1:1000; Cell Signaling Technology), and α-tubulin (1:30000–1:50000, Sigma-Aldrich). Densitometry of the bands obtained was performed using the ImageJ software (NIH, Bethesda, MD, USA).
3
Quantitative Western Blot Analysis of Protein Expression
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Total proteins were extracted from brain tissues and cells using protein lysis buffer (Cell Signaling Technologies, Danvers, MA). Equal amounts of protein (30 μg) were mixed with NuPAGE sample buffer (Invitrogen), boiled for 10 minutes at 70°C. Then the samples were separated by sodium NuPAGE 4% to 12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose i-blot gel transfer stack (Invitrogen) using i-blot system (Invitrogen). After the membrane had been blocked with Tris-buffered saline containing 5% non-fat dry milk, the membranes were treated with anti-STAT3, anti–phospho-STAT3 (p-STAT3; Cell signaling, 1:1,000), anti–cleaved caspase-3 (Cell signaling, 1:1,000), anti–BCL-XL (Cell signaling, 1:1,000), anti-BCL2 (Abcam, Cambridge, MA, 1:1,000), anti-Interleukin 6 (IL-6; Abcam, 1:1,000), anti-IL6R (Thermo Fisher Scientific, Rockford, IL, USA, 1:1,000), anti-ABCC4 (Cell signaling, 1:1,000), anti-Survivin (Abcam, 1:5,000), and anti–β-actin (Sigma-Aldrich, 1:5,000). The blots were developed with enhanced chemiluminescence reagent (Invitrogen) and were exposed to film. The blot densities of the proteins were normalized to the levels of internal β-actin expression and were represented as the relative intensity values.
4
Evaluating Signaling Pathways in Cell Viability
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LIQ was purchased from Shang Hai Winherb Medical Science CO., Ltd. (White crystalline powder, purity >98%). LPS was purchased from Sigma-Aldrich (St. Louis, MO, United States). Compound C (S7840) was purchased from Selleck (Shanghai, China). Primary antibodies including anti-mTOR (T-mTOR, 2983), anti-phospho-mTOR (p-mTOR, 2971), anti-GAPDH (2118), anti-Bax (2772), anti-Bcl-2 (2870), anti-phospho-JNK (p-JNK, 4468), anti-JNK (T-JNK, 9258), anti-phospho-ERK (p-ERK, 4370), anti-ERK (T-ERK, 4695), anti-phospho-p38 (p-p38, 4511), anti-p38 (T-p38, 9212), anti-phospho-AKT (p-AKT, 4691), anti-AKT (T-AKT, 4060), anti-phospho-GSK3β (p-GSK3β, 9323), anti-GSK3β (T-GSK3β, 9315), anti-phospho-STAT3 (p-STAT3, 9136), and anti-STAT3 (T-STAT3, 9139) were purchased from Cell Signaling Technology. Primary antibodies, including anti-phospho-AMPKα2 (p-AMPKα2, ab109402), anti-AMPKα2 (T-AMPKα2, ab3760), anti-phospho-IκBα (ab133462), anti-IκBα (ab7217), anti-phospho-p65 (ab194726), and anti-p65 (ab16502) were obtained from Abcam (Cambridge, United Kingdom). Secondary antibodies were obtained from LI-COR Biosciences. The cell counting kit 8 (CCK-8) was purchased from Dojindo Molecular Technologies (Rockville, MD, United States), and the Cu/Zn-SOD, Mn-SOD and NADP+/NADPH assay kits were purchased from Beyotime (Shanghai, China).
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