The expression pattern of Pol ι and ETS‐1 in human tissue samples was analyzed using immunohistochemistry. Tumor tissue sections were deparaffinized and heat‐treated with citrate buffer, pH 6.0, for 5 min as an epitope retrieval protocol. The tissue sections were then exposed to 0.03% hydrogen peroxide for 5 min to block endogenous peroxidase activity followed by incubation with Pol ι antibody (Proteintech) and ETS‐1 antibody (Abcam) diluted at 1:100 for 2 h at 37°C. HRP‐conjugated anti‐mouse/rabbit antibody was then added for 1 h and the color was developed using 3‐3′‐diaminobenzidine. Following washing, the sections were counterstained with hematoxylin, washed and dipped briefly in a water bath containing drops of ammonia, prior to dehydration and mounting in Diatex. The stained sections were analyzed and scored using a Leica Microscope (Leica, Wetzlar, Germany). Immunohistochemical scoring was based on the intensity and average percentage of positive cells. The staining intensity was scored with “1” (negative or weakly positive), “2” (moderately positive) and “3” (strongly positive). The average percentage of positive cells was scored as: 1 (<25%), 2 (25–50%), 3 (50–75%) and 4 (>75%).
Ets 1 antibody
Manufactured by Abcam
Sourced in United Kingdom
Ets-1 antibody is a laboratory reagent used to detect and quantify the Ets-1 protein in various biological samples. Ets-1 is a transcription factor that plays a crucial role in regulating gene expression during cellular processes such as cell growth, differentiation, and development.
Automatically generated - may contain errors
Lab products found in correlation
Chip it high sensitivity kit, by Active Motif (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Biotin 3 end dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Ez chip kit, by Merck Group (1 mentions)
Normal rabbit igg, by Beyotime (1 mentions)
4 protocols using ets 1 antibody
1
Immunohistochemical Analysis of Pol ι and ETS-1
2
Ets-1 Binding Site Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formaldehyde cross-linking and ChIP were performed using the ChIP-IT High Sensitivity Kit (Active Motif, Carlsbad, CA, USA), following the manufacturer’s instructions. Briefly, after test exposure, HMEC-1 were fixed for 10 min with complete cell fixation solution containing 37% formaldehyde, and sonicated to generate 500–800 bp DNA fragments. Immunoprecipitation was performed with protein G agarose beads and 4 μg of Ets-1 antibody (Abcam, Cambridge, UK). Chromatin extracts were incubated with the antibodies at 4 °C overnight under mild shaking, and ChIP DNA was eluted according to the manufacturer’s instructions. Precipitated DNA was purified and amplified by PCR with specific TF primers positive or negative for the presence of the Ets-1 binding site. PCR amplifications were performed with the primers listed in the Supplementary Material (Table S3) .
3
Ets-1 Transcription Factor Binding Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All primer sequences are provided in the Supplementary Material . Oligonucleotide probes were labeled using a Biotin 3’ End DNA Labeling Kit (Thermo Scientific, Darmstadt, Germany). Nuclear extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Waltham, MA, USA). The probe for Ets-1 used in EMSA (5′-TGGGCAAAGCATCCGGGAAATGCC-3′) corresponds to the TF promoter region: −498 to −475 bp. The binding mixture contained 5 µg nuclear extract, 20 fmol labeled double-stranded probe, 1 µg poly-dI/dC, and 1X reaction buffer. Incubation was performed at room temperature for 30 min. Protein–DNA complexes were then analyzed by electrophoresis in 6% non-denaturing polyacrylamide gels and visualized using a LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Darmstadt, Germany). In supershift experiments, nuclear extracts were incubated with Ets-1 antibody (Abcam, Cambridge, UK) before adding the biotin-labeled probe.
4
ChIP Assay for ETS1 Transcription Factor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed with EZ ChIP kit (Millipore, Billerica, MA, USA) and ETS1 antibody (ab124282, Abcam). The primers used in the PCR analysis of this assay were: 5′-TGGTCAGAACCCCCTGATGA-3′ and 5′-TCCACAGCAAACATGTGGAGA-3′. Normal rabbit IgG (Beyotime, Shanghai, China) was used as a negative control in the immunoprecipitation.
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!