We collected 160 human epithelial ovarian cancer samples with accompanying patient follow-up information between January 2010 and January 2015 in the Department of Pathology of Peking Union Medical College Hospital. Follow-up was performed until January 1, 2020. Pathological diagnoses were reconfirmed by a pathologist. The project was approved by the Ethics Committee (Peking Union Medical College Hospital), and informed consent was obtained from patients or their family members. The details of immunohistochemical staining (IHC) and scoring were described previously (Li et al., 2010 (link); Zhang et al., 2015 (link)). The following antibodies were used: anti-EZH2, 1:200, Abcam ab191080; anti-CYP27B1, 1:500, Abcam ab206655. The intensity of immunostaining was scored as follows: 1 +, weak; 2 +, moderate; 3 +, strong; or 4 +, very strong. The area of positive staining in each microscopic field was scored as follows: 1 +, 0–25%; 2 +, 25–50%; 3 +, 50–75%; or 4 +, 75–100%. A final score between 5 and 80 was obtained by multiplying the product of the two scores by 5. A final score between 0 and 42 was considered “low expression” and a final score between 43 and 80 considered “high expression.” All pathological diagnoses were confirmed in a blinded manner by three expert pathologists.
Ab191080
Manufactured by Abcam
Sourced in United States
Ab191080 is a recombinant anti-Rabbit IgG (H+L) monoclonal antibody. The antibody is designed for use in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab6002, by Abcam (5 mentions)
Ab1791, by Abcam (4 mentions)
Ab192985, by Abcam (4 mentions)
Ab15580, by Abcam (4 mentions)
Gtx121246, by GeneTex (4 mentions)
Ab202975, by Abcam (2 mentions)
Ab138490, by Abcam (2 mentions)
Ab195121, by Abcam (2 mentions)
Ab181136, by Abcam (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Vinculin, by Merck Group (2 mentions)
Ab230445, by Abcam (2 mentions)
Sc 7392, by Santa Cruz Biotechnology (2 mentions)
Baf a1 s1413, by Selleck Chemicals (2 mentions)
Sc 17775, by Santa Cruz Biotechnology (2 mentions)
Sc 373822, by Santa Cruz Biotechnology (2 mentions)
Vegfc, by ABclonal (2 mentions)
F3165 1mg, by Merck Group (2 mentions)
Myc tag, by Proteintech (2 mentions)
Ha tag, by Proteintech (2 mentions)
23 protocols using ab191080
1
Immunohistochemical Evaluation of Ovarian Cancer Markers
2
Comprehensive Epigenetic Regulatory Analysis
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Rabbit anti‐KDM2B (09‐864; Millipore), anti‐EZH2 (ab191250 and ab191080; Abcam), anti‐LaminB1 (ab16048; Abcam), and goat‐anti rabbit HRP (CST) antibodies were used for western blots or immunohistochemistry (IHC). H3K36me2 (ab9049; Abcam) and RNA pol II (ab193468; Abcam) antibodies were used in a ChIP assay. Real‐time PCR primers and oligos used for shRNA cloning are listed in Table 1 . The oligos of hsa‐let‐7b‐5p‐inhibition(21257‐1)‐a and hsa‐let‐7b‐5p‐inhibition(21257‐1)‐b were used for cloning the let‐7b inhibitor sequence into the GV280 vector, and the oligos of hsa‐let‐7b(16144‐1)‐P1 and hsa‐let‐7b(16144‐1)‐P2 were used for cloning the let‐7b mimic sequence into the GV369 vector. Primers of KDM2B(21416‐11)‐P1 and KDM2B(21416‐11)‐P2 were used to amplify KDM2B coding sequences, and the 4062bp PCR product was cloned into the Age I/Age I site of GV358 vector. The sequences of shKDM2B, shEZH2, and shNC are as follows: TTCTTCAAACGCTGTGGAA, GAAATCTTAAACCAAGAAT, and TTCTCCGAACGTGTCACGT, respectively.
3
Protein Quantification and Western Blot Analysis
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The total protein was extracted from tissues and cells, and the protein quantification was performed by bovine serum albumin protein kit (Thermo Scientific, Rockford, IL, USA). SDS polyacrylamide gel electrophoresis (10%) was prepared and each well was added with 25 μg protein sample. After electrophoresis, protein was transferred into the polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA), which was blocked by 5% skimmed milk powder for 1 h, washed three times with phosphate-buffered saline with Tween, and incubated with EZH2 antibody (ab191080, 1:1000), H3K27me3 antibody (ab6002, 1: 1000), ZC3H12D antibody (ab100862, 1:1000), histone H3 antibody (ab1791. 1:1000), and glyceraldehyde phosphate dehydrogenase (ab9485, 1:2000) (all from Abcam) overnight, then incubated with secondary antibody labeled by enzyme (Abcam) for 1 h. The membrane was developed and exposed by enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL, USA). The gray value was analyzed by ImageJ software.
4
Comprehensive Antibody and Inhibitor Panel
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Primary antibodies used in this study are as follows: UTX (33510S, Cell Signaling Technology; GTX121246, GeneTex), COP1 (A300-894A, Bethyl), CUL1 (SC-17775, Santa Cruz Biotechnology), CUL2 (A5308, Abclonal), CUL3 (2759, Cell Signaling Technology), CUL4A (AB60215a, Abgent), CUL4B (A6198, Abclonal), CUL5 (SC-373822, Santa Cruz Biotechnology), EMP1 (ab230445 and ab202975, Abcam), AUTS2 (25,001–1-AP, Proteintech), Ki67 (ab15580, Abcam), EZH2 (ab191080, Abcam), VEGFC (A2556, Abclonal), Caspase-9 (A0281, Abclonal), Bcl2 (15,071, Cell Signaling Technology), DDB2 (ab181136, Abcam), DCAF7 (ab138490, Abcam), DCAF13 (ab195121, Abcam), H3K27me3 (ab192985, Abcam; A2363, Abclonal), H3 (ab1791, Abcam), P27 (610,241, BD), HA-Tag (51,064–2-AP, Proteintech; SC-7392, Santa Cruz Biotechnology), Flag-Tag (F7425-0.2MG, Sigma; F3165-1MG, Sigma), GFP (66,002–1-Ig, Proteintech), Myc-Tag (16,286–1-AP, Proteintech), α-tubulin (SC-23948, Santa Cruz Biotechnology), and Vinculin (V4505, Sigma). The chemical inhibitors MG132 (S2619), MG101 (S7386), MLN4924 (S7109), Baf-A1 (S1413), and GSK126 (S7061) were purchased from Selleck.
5
Immunofluorescence Analysis of EZH2 and H3K27me3
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After fixation and permeabilization, the BMSCs were divided into two groups. Group 1 received rabbit anti-EZH2 (ab191080, Abcam) at a 1/250 dilution, followed by goat anti-rabbit IgG (AlexaFluor® 488) (ab150077, Abcam) secondary antibody (green) at a 1/200 dilution. Group 2 received mouse anti-tri-methyl-histone H3(lys27) (ab6002, Abcam) at a 1/1000 dilution, followed by goat anti-mouse IgG H&L (Alexa Fluor® 594) (ab150120) at a 1/1000 dilution Resist (red). The nuclear counterstain was DAPI (blue).
6
EZH2 Immunoprecipitation and RIG-I Detection
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SMCs were lysed using cell lysis solution on ice for 30 min, after which the cell lysate was collected into a 1.5 mL Eppendorf tube and centrifuged at 12,000×g and 4 °C for 15 min, with the supernatant collected. Then 50 μL of protein A and protein G beads were extracted to the tube and suspended to form a mixture of 100 μL beads, 10 μL of which was added to the cell lysate with EZH2 antibody (ab191080, 1:1000, Abcam) and incubated at 4 °C overnight for coupling. After IP reaction, centrifugation was carried out at 3000 rpm and 4 °C for 3 min, with the agarose beads to the bottom of the tube. At last, 15 μL of 2 × sodium dodecyl sulfate (SDS) loading buffer was added to the precipitate, and boiled for 5 min. After cooling, the precipitate was analyzed by Western blot analysis to detect the expression RIG-I.
7
Immunohistochemical Profiling of UTX and COP1
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IHC staining was performed with indicated antibodies using the standard protocol. The TMA was incubated with an anti-UTX antibody (1:400 dilution, 33510S, Cell Signaling Technology) and an anti-COP1 antibody (1:100 dilution, A300-894A, Bethyl). Mouse tissues were incubated with anti-UTX antibody (1:100 dilution, GTX121246, GeneTex), anti-COP1 antibody (1:100 dilution, A300-894A, Bethyl), Ki67 (1:100 dilution, ab15580, Abcam), anti-EZH2 antibody (1:200 dilution, ab191080, Abcam), and anti-H3K27me3 antibody (1:100 dilution, ab192985, Abcam; 1:100 dilution, A2363, Abclonal), respectively, as indicated. The IHC stained tissue sections were scored by two independent pathologists blinded to the clinicopathological features. The proportion of staining was graded as 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%) based on the percentages of the positive staining areas vs. the whole area. The staining intensity was graded as 0 (negative), 1 (weak), 2 (medium,) or 3 (strong). The immunoreactivity score (IRS) was calculated by multiplying the score of staining proportion with staining intensity. IRS ≤ 4 was considered as low, and > 4 was considered as high.
8
Immunohistochemical Analysis of EZH2 in Wilms' Tumor
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Formalin-fixed, paraffin embedded (FFPE) sections (4 μm) were deparaffinized in xylene and rehydrated to water through graded alcohols. Endogenous peroxidase was blocked with 1% H2O2 (Sigma-Aldrich) diluted in PBS pH 7.4 (Applichem) for 20 min. Heat induced epitope retrieval (HIER) was performed with Target Retrieval Solution pH 9.0 (DAKO) and 0.2 % Triton-X100 (Sigma) in a pressure cooker at 120°C for 20 min. Sections were incubated with primary antibody EZH2 1:400 (ab191080, Abcam) diluted in PBS containing 5% normal goat serum (Jackson Immuno Research) for 60 min. Staining was visualized using BrightVision Poly-HRP-Anti rabbit RTU (AH Diagnostics) for 30 min, followed by incubation with DAB (Liquid DAB+ Substrate Chromogen System, DAKO) for 5 min, and counterstained with Mayers Htx (Histolab) for 30 sec. Slides were mounted with Faramount Aqueous Mounting Medium (DAKO). A tissue microarray with cores from the different histological compartments in totally 33 primary WTs [32 (link)] was used to investigate EZH2-protein expression.
9
Quantifying RUNX3 and EZH2 Expression
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Antibodies used to quantify RUNX3 expression (ab224641) and EZH2 expression (ab191080) were purchased from Abcam (Cambridge, UK). The expression of these proteins was determined using immunohistochemistry. Specimens obtained from preoperative biopsy tissue were cut into 5 μm sections. The sections were examined under a microscope. Five fields were evaluated, and the proportion of positive cells was counted, regardless of staining intensity. RUNX3 and EZH2 expression were divided into two groups: high expression group and low expression group. In the high expression group, at least 50% of the nuclei were positive whereas in the low expression group, the nucleus was less than 50% positive.
10
Western Blot Analysis of Cancer Biomarkers
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This study utilized RIPA buffer (Beyotime Institute of Biotechnology) that contained protease K inhibitor for extracting total proteins, which were then separated through 12% SDS-PAGE, followed by transfer onto PVDF membrane. Subsequently, membrane was immersed within 5% defatted milk under ambient temperature for a 2 h, followed by overnight incubation under 4 °C using corresponding primary antibodies: EZH2 (1:500, ab191080; Abcam, Cambridge, MA, USA), p-STAT3 (1:2000, ab76315; Abcam, Cambridge, MA, USA), STAT3 (1:1000, ab68153; Abcam, Cambridge, MA, USA), Mcl-1 (1:1000, ab32087; Abcam, Cambridge, MA, USA) and p-Bcl-2 (1:1000, ab218123; Abcam, Cambridge, MA, USA), H3K27me3 (1:1000, ab6002; Abcam, Cambridge, MA, USA) with GAPDH being the loading reference. Then, the membrane was subject to secondary antibody incubation (1:5000, ab96899; Abcam, Cambridge, MA, USA). Protein band visualization was performed using Chemidoc EQ system (Bio-Rad Laboratories, Inc.).
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