ChIP assay was performed using a ChIP kit (Abcam) using an anti-Twist1 antibody (Abcam) according to the manufacturer’s instructions.33 (link) OA-FLSs were cultured to collect chromatin, which was immunoprecipitated with either Twist1 or control antibodies. Extracted DNA was amplified by PCR using primer pairs specific for the E box in the promoter region of miR-10a.
Anti twist1 antibody
Manufactured by Abcam
Sourced in United States
Anti-TWIST1 antibody is a laboratory tool used to detect and study the TWIST1 protein. TWIST1 is a transcription factor involved in various cellular processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunofluorescence to identify and analyze the TWIST1 protein in biological samples.
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Lab products found in correlation
Anti snail2, by Cell Signaling Technology (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (2 mentions)
Anti zeb1, by Santa Cruz Biotechnology (2 mentions)
Anti zeb2, by Merck Group (2 mentions)
Anti ha and anti α tubulin antibodies, by Merck Group (2 mentions)
Anti e cadherin, by BD (2 mentions)
Chip kit, by Abcam (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Anti vhl antibody, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Dmire2 microscope, by Leica (1 mentions)
Ccd camera dfc420, by Leica (1 mentions)
Dab substrate chromogen system, by Agilent Technologies (1 mentions)
Twist1, by Abcam (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
6 protocols using anti twist1 antibody
1
ChIP Assay for Twist1 Binding
2
Western Blot Analysis of EMT Markers
3
Proximity Ligation Assay for TWIST1-CBLC Interaction
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Proximity ligation assay was performed with the Duolink PLA kit (#Duo94104, ThermoFisher) according to the manufacturer’s instructions. HEK-293 T cells were fixed and incubated overnight with anti-TWIST1 antibody (#ab50518, Abcam; Cambridge, MA, USA) and anti-CBLC antibody (#F-2, Santa Cruz; CA, USA). After multiple washings, cells were incubated successively with PLA probes, ligation solution and amplification solution at 37℃. Cover-slips were mounted, and the images were photographed using Leica TCS SP5 confocal microscope (Leica Microsystems; Mannheim, Germany).
4
Immunohistochemical Analysis of VHL and Twist1
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Tissue sections were deparaffinized twice by xylene and then hydrated. Hydrogen peroxide (0.6%) was used to eliminate endogenous peroxidase activity. The sections were blocked with goat serum in Tris-buffered saline for 30 min. Sections were then incubated with anti-VHL antibody (Abcam, 1:100) and anti-Twist1 antibody (Abcam, 1:500) overnight at 4 °C. Secondary antibody was then applied and incubated at 37 °C for 1 hour. Sections were developed with diaminobenzidine and stopped with water. Photographs of representative fields were captured using a Leica CCD camera DFC420 connected to a Leica DMIRE2 microscope (Leica Microsystems Imaging Solutions, Cambridge, UK). Quantification of immunoreactivity was performed on digitally captured color images saved as TIFF files and analyzed using Image-Pro plus 6.0 (Media Cybernetics, Rockville, Maryland, USA). These methods were performed in accordance with the approved guidelines and regulations from the Research Ethics Committee of Huashan Hospital, Fudan University.
5
Immunoblotting Antibody Validation Protocol
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Immunoblotting was carried out according to standard procedures.12 (link) The following primary antibodies were used in this study: anti-EGFP (ImmunoKontact, UK; catalog number 210-PS-1GFP), anti-ZEB1 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-25388); anti-SNAIL2 and anti-Cyclin D1 (Cell Signaling Technology, Danvers, USA; 9585 and 2978) anti-ZEB2 (in-house12 (link)); anti-HA and anti-α-Tubulin antibodies from Sigma-Aldrich (St Louis, MO, USA; H3663 and T5168); anti-TWIST1 antibody from Abcam (Cambridge, MA, USA; AB50887); and anti-E-cadherin (BD Bioscience, San Jose, USA; 610181).
6
Immunoblot and Immunohistochemical Analysis of TWIST1
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Immunoblot analysis was performed as previously described [25] using anti-TWIST1 antibody (1:1000 dilution; Abcam) and anti-GAPDH antibody (1:1000 dilution; Cell Signal Technology).
For the immunohistochemistry (IHC) analysis, the excised tumors were xed with 4% paraformaldehyde and sectioned into 6-µm sections. Brie y, after depara nization, antigen retrieval and quenching of endogenous peroxidase activity, the slides were incubated with the primary antibody (TWIST1, 1:200 dilution, Abcam) overnight at 4 ℃ in a humid chamber. A negative control was obtained by replacing the primary antibody with a normal IgG. Thereafter, slides were incubated with the secondary antibody conjugated to horseradish peroxidase (HRP). Antibody binding was visualized using the DAB + Substrate Chromogen System (DAKO). Finally, sections were counterstained with hematoxylin, dehydrated, cleared, and photographed.
The immunohistochemistry scoring was performed by two independent observers. Unequivocal nuclear staining pattern for TWIST1 was interpreted based on the intensity as negative, weak, moderate and strong. Moderate to strong nuclear staining was considered to be positive reaction. The distribution of TWIST1 was scored as follows: negative (less than 50% of the cells being positive) and positive (where more than 50% of the cells were positive).
For the immunohistochemistry (IHC) analysis, the excised tumors were xed with 4% paraformaldehyde and sectioned into 6-µm sections. Brie y, after depara nization, antigen retrieval and quenching of endogenous peroxidase activity, the slides were incubated with the primary antibody (TWIST1, 1:200 dilution, Abcam) overnight at 4 ℃ in a humid chamber. A negative control was obtained by replacing the primary antibody with a normal IgG. Thereafter, slides were incubated with the secondary antibody conjugated to horseradish peroxidase (HRP). Antibody binding was visualized using the DAB + Substrate Chromogen System (DAKO). Finally, sections were counterstained with hematoxylin, dehydrated, cleared, and photographed.
The immunohistochemistry scoring was performed by two independent observers. Unequivocal nuclear staining pattern for TWIST1 was interpreted based on the intensity as negative, weak, moderate and strong. Moderate to strong nuclear staining was considered to be positive reaction. The distribution of TWIST1 was scored as follows: negative (less than 50% of the cells being positive) and positive (where more than 50% of the cells were positive).
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