The expression vectors for WT and mutant ERβ were described previously [34 (link)]. The plasmid for p300 expression was kindly provided by Dr. Zhi-Min Yuan (Harvard T.H. Chan School of Public Health). Cell lines were originally purchased from the American Type Culture Collection and cultured per their instructions.17-β-estradiol (E2), DPN, and MG132 were obtained from Tocris, Inc. S-equol was generously provided by Ausio Pharmaceuticals. The following antibodies were purchased commercially: anti-Flag M2 (A8592 and F3165, Sigma-Aldrich), anti-ERβ for immunoblotting (14C8, GeneTex; 9.88, Abcam), anti-ERβ for immunoprecipitation (IP) (EPR3777, Novus), anti-p300 (sc-584, Santa Cruz Biotechnology Inc.), anti-GAPDH (G9295, Sigma-Aldrich), anti-FLAG-HRP (A8592, Sigma-Aldrich), anti-Flag M2 agarose (A2220, Sigma-Aldrich), and anti-Ki67 (GTX16667, GeneTex). The rabbit polyclonal anti-pY36 antibody was raised as previously described [34 (link)]. Oligonucleotides si-Con (non-targeting) (L-001810-10), si-p300 (L-003486-00), ON-TARGETplus smart pool siRNA duplexes were purchased from Dharmacon.
Anti ki67
Manufactured by GeneTex
Sourced in United States
Anti-Ki67 is a laboratory equipment product that is used to detect the presence and expression of the Ki67 protein, which is a marker of cell proliferation. The Anti-Ki67 product is designed to be used in various biological and medical research applications.
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Lab products found in correlation
Anti flag m2 agarose, by Merck Group (1 mentions)
Anti p300, by Santa Cruz Biotechnology (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Anti desmin, by Thermo Fisher Scientific (1 mentions)
Anti pcna, by BioLegend (1 mentions)
Alexa fluor 594 and alexa fluor 488 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti desmin antibody, by Thermo Fisher Scientific (1 mentions)
Anti α sma antibody, by Abcam (1 mentions)
Anti rfp, by Rockland Immunochemicals (1 mentions)
Anti cd44, by Bio-Rad (1 mentions)
Anti hnf4α, by Abcam (1 mentions)
Anti hoxa10, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 or 568, by Thermo Fisher Scientific (1 mentions)
Anti myod, by Proteintech (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
8 protocols using anti ki67
1
Characterization of Mutant Estrogen Receptor-Beta
2
Immunohistochemical Evaluation of CREPT and Ki-67
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The paraffin‐embedded tissues were sliced into 3‐μm sections and deparaffinized, then the slides were boiled in 10 mmol/L citrate buffer for antigen retrieval and blocked with 10% goat serum. The slides were then incubated with primary anti‐CREPT (1:200; GeneTex) or anti‐Ki‐67 (1:200; GeneTex) antibodies overnight. The same concentration of antigen‐specific antibody (Kangwei) was used as negative control. After washing with PBS, the tissue sections were incubated with EnVision HRP (Kangwei, China) as the secondary antibody. Finally, the DAB Elite kit (Zhongshan, China) was used for chemiluminescence analysis. All stained sections were examined by two independent investigators who were blinded to the clinical features and outcomes. The immunohistochemical (IHC) staining scores were based on the following criteria: (i) the percentage of positive cells (0, ≤5%; 1, 6%‐25%; 2, 26%‐50%; 3, 51%‐75%; and 4, >75%); (ii) the staining intensity (0, no color; 1, yellow; 2, brown; and 3, tan); and (iii) the two grades were multiplied together and specimens were assigned to one of four levels: 0, negative (−); 1‐4, weakly positive (+); 5‐8, moderately positive (++); and 9‐12, strongly positive (+++).8 , 15 To investigate the correlation of protein expression, (−) and (+) were considered as low expression, and (++) and (+++) were considered as high expression.
3
Immunohistochemical and Immunofluorescent Analysis of Liver Markers
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For immunohistochemical analysis, mouse liver sections were deparaffinized, rehydrated and incubated with anti-desmin antibody (1:200, Thermo Fisher Scientific, Fremont, CA), anti-α-SMA antibody (1:200, Abcam, Cambridge, MA) or anti-F4/80 (1:200, eBioscience, San Diego, CA) and stained using DAKO Envision system (DAKO Corp., Carpinteria, CA). The area of positive staining was measured in low-power (x10) fields on each slide and quantified using NIH imaging software. For immunofluorescent staining, the sections were incubated with anti-HNE (1:200, Alpha Diagnostics, San Antonio, TX), anti-PCNA (1:200, Biolegend Inc., San Diego, CA), anti-desmin (1:200, Thermo Fisher Scientific, Fremont, CA), or anti-Ki67 (1:200, GeneTEX, INC. Irvine, CA) antibodies and Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (1:200, Invitrogen, Carlsbad, CA) and imaged with fluorescent microscopy.
4
Immunohistochemical Profiling of Cell Markers
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The following antibodies were used in the experiments: anti‐CD44 (AbD Serotec); anti‐Sox9 and anti‐K19 (Santa Cruz Biotechnology), anti‐F4/80 (Caltag), anti‐Ki67 (Gene Tex), anti‐HNF4α (Abcam), and anti‐RFP (Rockland).
5
Immunohistochemical Analysis of HOXA10, BCL2, and Ki67
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The tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol followed by epitope retrieval in citrate buffer (pH = 6.0). HOXA10, BCL2, and Ki67 expressions were detected using antibodies anti‐HOXA10 (1:100, Santa Cruz, USA), anti‐BCL2 (1:100, CST, USA), and anti‐Ki67 (1:600; GeneTex), respectively. After the secondary antibody incubation with Envision System Plus‐HRP, the slides were rinsed and counterstained with Mayer's hematoxylin. Finally, sections were imaged and evaluated.
6
Immunohistochemical Analysis of Ki67 Expression
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After paraffin-embedded tissue was sliced, dewaxed, hydrated, and antigen-repaired, with additional blockage of endogenous peroxidase, anti-Ki67 (Genetex, GTX 16667) was added, and the samples were refrigerated at 4°C overnight. Polymer enhancer was added dropwise for 20 min at room temperature, and a biotin-labeled secondary antibody was added dropwise. The samples were then incubated at room temperature for 30 min. DAB was used as the color body while hematoxylin was used as the color former. Finally, a conventional dehydrated and transparent neutral gum seal was used. The degree of immunostaining of the paraffin-embedded sections was assessed and scored by two independent pathologists who were blinded to the histopathological features and patient data. The score was determined by combining the proportion of positively stained tumor cells and staining intensity. Specific scoring rules were based on previous research.
7
Immunofluorescence Imaging of Muscle Stem Cells
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PMSCs were grown on confocal dishes and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing with PBS, cells were permeabilized and blocked with 0.3% Triton X-100 and 3% BSA in PBS for 1 h at room temperature. Cells were then incubated with primary antibodies against anti-Ki67 (Monoclonal, 1:100, GeneTex, Irvine, CA, USA), anti-MyoD (Polyclonal, 1:200, Proteintech, Rosemont, IL, USA), and anti-Pax7 (Monoclonal, 1:50, DSHB, Iowa, IA, USA) at 4 °C overnight. After washing with PBS, primary antibodies were visualized with fluorescently labeled secondary antibodies (Alexa Fluor 488 or 568; Molecular Probes, Eugene, OR, USA). Cells were then stained with 4’-6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Fluorescence images were collected using a super-resolution confocal laser scanning microscope (SR-CLSM) installed in the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University.
8
Evaluating Microscopic Metastasis in Neuroblastoma
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To evaluate microscopic metastasis, harvested organs were xed in a 4% paraformaldehyde solution at 4°C for 48 h, embedded in para n, and sectioned into 3 µm-thickness at the maximum cut surface. After staining with hematoxylin and eosin (H&E), the average number of metastatic foci and nuclear divisions in each of 20 randomly selected high-power microscopic elds were determined at 400 × magni cation.
Para n-embedded liver neuroblastoma blocks were used for immunohistochemical analysis. The samples were depara nized, heated in citrate buffer solution (pH 6.0) or EDTA (pH 8.0) to unmask antigens, and washed with PBS. The primary and secondary antibodies used were anti-Ki-67 (1:100, Gene Tex, North America, USA) or cleaved caspase-3 (1:100, Cell Signaling Technology, Danvers, USA), and Simple Stain MAX PO (1:100, Nichirei Biosciences Inc., Tokyo, Japan), respectively. After slide pictures were taken using a TOCO virtual slide scanner (PATH IMAGING Inc., Tokyo, Japan), they were assessed with the help of Image J (National Institutes of Health, Bethesda, Maryland, USA) [16, 17] (link). The tumor number and metastatic foci per liver area were counted using Ki-67 staining. Ki-67-positive cells per tumor area were also evaluated in the 16-20 foci. The apoptotic cell number was determined by cleaved caspase-3 positivity per tumor area in the 16-20 foci.
Para n-embedded liver neuroblastoma blocks were used for immunohistochemical analysis. The samples were depara nized, heated in citrate buffer solution (pH 6.0) or EDTA (pH 8.0) to unmask antigens, and washed with PBS. The primary and secondary antibodies used were anti-Ki-67 (1:100, Gene Tex, North America, USA) or cleaved caspase-3 (1:100, Cell Signaling Technology, Danvers, USA), and Simple Stain MAX PO (1:100, Nichirei Biosciences Inc., Tokyo, Japan), respectively. After slide pictures were taken using a TOCO virtual slide scanner (PATH IMAGING Inc., Tokyo, Japan), they were assessed with the help of Image J (National Institutes of Health, Bethesda, Maryland, USA) [16, 17] (link). The tumor number and metastatic foci per liver area were counted using Ki-67 staining. Ki-67-positive cells per tumor area were also evaluated in the 16-20 foci. The apoptotic cell number was determined by cleaved caspase-3 positivity per tumor area in the 16-20 foci.
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