For IHC staining, paraffin-embedded tumour slides were deparaffinised, rehydrated and incubated with primary antibodies overnight at 4 °C. Primary antibodies against AEG-1, E-cadherin, vimentin and VEGF (Abcam, UK) were used for detection. The slides were then incubated with a biotin-labelled secondary antibody (Zhongshan Bio Corp., China) for 1 h at room temperature and then with diaminobenzidine (Zhongshan Bio Corp., China). The results were assessed by measuring both the staining intensity and the number of positive cells. The intensity of a positive reaction was scored as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = intense staining. Staining was also scored on a scale of 0–3 according to the percentage of cells involved, where 0 = 0–5%; 1 = 6–25%; 2 = 26–50%; and 3 = 51–100% positive cells. The scores for the intensity and the percentage of positive cells were multiplied to calculate a weighted score for each case. A score of 0–3 was defined as low expression (−) and scores of 4–9 as high expression (+).
Aeg 1
Manufactured by Abcam
Sourced in United Kingdom, United States
AEG-1 is a lab equipment product manufactured by Abcam. It is designed to perform a core function within a laboratory setting, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or specific capabilities of the AEG-1 is not available.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
Nf κb p65, by Santa Cruz Biotechnology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Mmp 9, by Abcam (1 mentions)
Stat3, by BD (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gapdh antibodies, by Immunoway (1 mentions)
Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions)
P nf κb p65, by Santa Cruz Biotechnology (1 mentions)
6 protocols using aeg 1
1
Immunohistochemical Analysis of Tumor Biomarkers
2
Protein Expression Analysis in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in a RIPA buffer to obtain total proteins. Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated with primary antibodies overnight at 4°C and then with linked secondary antibodies. Primary antibodies were listed as followed: AEG-1 (Abcam), MMP-9 (Abcam), P-STAT3 (Tyr705) (Cell Signaling), STAT3 (BD Bioscience), NF-κB p65 (Santa Cruz), β-Actin (ZSJB-BIO).
3
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Western blot analysis and the whole-cell lysates were carried out as described in detail previously [19 (link)]. The blots were probed with AEG-1 (Abcam Co., Milton, UK), Bcl-2, Bax, cleaved-caspase-3, caspase-3, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38 MAPK, p38 MAPK (Cell Signaling Technology Co., Danvers, MA, USA) and GAPDH antibodies (ImmunoWay Co., Newark, DE, USA). The protein expression was visualized after extensive washing using the ECL (enhanced chemiluminescence) advanced detection kit (GE Healthcare Co., Buckinghamshire, UK) and quantified with the Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
4
Molecular Profiling of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested in logarithmic phase and lysed with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.4). Total protein and nucleus protein were extracted. Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with the primary antibody overnight at 4 °C, with a secondary antibody (Zhongshan Bio-Tech Co., Beijing, China) for 1 h at room temperature, and then scanned using an Odyssey infrared imaging system (LICOR, Lincoln, NE, USA). Primary antibodies against AEG-1, E-cadherin, N-cadherin, vimentin (Abcam, UK), VEGF, p-p38, p38, Caspase-3 and PARP (Cell Signaling Technology Inc., Danvers, MA, USA), c-Myc, NF-κB p-p65, NF-κB p65 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), and GADPH (KangChen Biotech, Shanghai, China) were used for western blotting analysis.
5
Quantitative Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein extracted from cells was isolated using RIPA lysis buffer with 1 mM PMSF and kept on ice for 10 min followed by 15 min of centrifugation at 12 000 rpm and 4 °C, and the protein con-centration was measured using the BCA protein assay kit. 40 ng of protein was separated by sodium dodecyl-sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat milk for 2 h, the membranes were incubated with the primary antibody against AEG-1 (1: 1000, Abcam, USA), MDR-1 (1: 1000, Abcam, USA) and GAPDH (1: 2000, Abcam, USA), overnight at 4 °C, and followed by subsequent incubation with the respective secondary antibodies for 2 h at room temperature. After washing the blots three times with TBST, they were visualized using electrochemiluminescence plus reagent. Finally, the intensities of these blots were quantified using Image Lab 3.0 software (Bio-Rad).
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, China). The protein concentrations were determined using the bicinchoninic acid assay (BCA). A total of 40 μg of proteins was electrophoresed via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were then blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were incubated with antibodies against AEG-1 (1:1000 dilution; Abcam, USA), Bcl-2 (1: 100 dilution), Bax (1: 100 dilution), caspase-3 (1: 100 dilution; Biosynthesis Biotechnology, China), PCNA (1: 500 dilution), Cyclin D1 (1: 200 dilution), Cyclin E (1: 200 dilution), PARP (1: 500 dilution; Santa Cruz Biotechnology, USA), followed by incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Institute of Biotechnology) at a 1:5000 dilution for 1 h at 37°C. Chemiluminescence was then produced by chemiluminescence substrate (Thermo Scientific Pierce) and subsequently exposed to X-film and scanned into the computer. Protein expression levels from each group were determined via gray scale analysis using Image J software. The β-actin served as an internal control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!