To immunostain iPSCMs, cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 10 min, permeabilized with 0.1% Triton X-100 (Wako) for 5 min, and blocked with PBS containing 5% goat serum (Wako) for 1 hour at room temperature. After the samples were incubated with primary antibodies overnight at 4°C, they were incubated with Alexa Fluor–conjugated secondary antibodies for 1 hour at room temperature. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). Images were captured using a confocal microscope (Carl Zeiss, LSM 880) and analyzed using ZEN (Carl Zeiss). The primary antibodies were Lamin A/C (4C11) (1:400; Cell Signaling Technologies, #4777), TNNT2 (13-11) (1:500; Thermo Fisher Scientific, #MA5-12960), and TEAD1 (1:100; Abcam, Cambridge, UK, #ab133533).
Tead1
Manufactured by Abcam
Sourced in United States
TEAD1 is a transcriptional enhancer factor that is involved in the regulation of gene expression. It is a member of the TEA domain family of transcription factors.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Abcam (3 mentions)
Tyramide superboost kit, by Thermo Fisher Scientific (2 mentions)
Taz wwtr1, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Fujifilm (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Goat serum, by Fujifilm (1 mentions)
Lamin a c 4c11, by Cell Signaling Technology (1 mentions)
Primary antibody for elf3, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Tead2, by Abcam (1 mentions)
Lats1, by Abcam (1 mentions)
Lats2, by Abcam (1 mentions)
Tead4, by Abcam (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
8 protocols using tead1
1
Immunostaining of Induced Pluripotent Stem Cell-Derived Cardiomyocytes
2
Immunohistochemical Analysis of FZD5, TEAD1, ELF3, and SNAI2
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Paraffin-embedded sections were deparaffinized and rehydrated. Sections were in turn added endogenous peroxidase blockers, normal non-immune animal serum, primary antibody, biotin–conjugated secondary antibody, streptomycin antibiotic-peroxidase solution, freshly prepared DAB. Then sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared and mounted. Primary antibodies for FZD5 (1:100) and TEAD1 (1:200) was purchased from Abcam. Primary antibody for ELF3 (1:200) was provided by Thermo Scientific. Primary antibody for SNAI2 (1:200) were obtained from Cell Signaling Technology. H-Score system (range: 100–400) was used to assess the protein expression [20 (link)].
3
Immunostaining Protocols for Islet Analysis
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Immunostaining was performed as previously described.5 (link) Primary antibodies used were TEAD1 (rabbit polyclonal, 1:200; Abcam), Ki67 (rabbit polyclonal, 1:50; Abcam), BRDU (rat polyclonal, 1:1,000; Abcam), TAZ (WWTR1) (mouse monoclonal, 1:100, Cell signaling). Tyramide SuperBoost Kits (Thermofisher) were used for TAZ (WWTR1) staining according to the manufacturer’s instructions in islet. The images were taken by the Nikon confocal microscope.
4
Immunostaining Protocols for Islet Analysis
5
Western Blot Analysis of Hippo Pathway Proteins
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A total of 0.05 μg tissue samples were collected using RIPA lysis buffer (Millipore, Billerica, MA, USA) containing protease inhibitors (Sigma, Shanghai, China). Protein samples were centrifuged and collected. The BCA method (Pierce, Rockford, IL, USA) was used to determine the concentration according to manufacturer’s protocol (Beyotime, Nanjing, Jiangsu, China). Subsequently, proteins as well as loading buffer were heated, after which proteins were separated by 10% SDS electrophoresis and transferred to polyvinylidene fluoride membranes (GVS Technology Co., Ltd., Suzhou, China). After blocked with milk at room temperature for one hour, membranes were incubated in a 1:1000 solution of primary antibodies (MST, MST2, LATS1, LATS2, YAP1, TAZ, TEAD1, TEAD2, TEDA3, TEAD4, GAPDH; Abcam, Cambridge, MA, USA) at 4 °C overnight. Subsequently, membranes were incubated with the secondary antibody at room temperature for one hour and observed using the ECL method (Pierce, Rockford, IL, USA).
6
Western Blot Analysis of Exosomal Proteins
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Heart tissues, exosomes, and cells were lysed using RIPA buffer (Beyotime, Jiangsu). Protein concentration was measured by the BCA method (Thermo Fisher Scientific, USA). Equal amounts of protein were loaded on SDS-PAGE gels, separated, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Then, the membranes were incubated with primary rabbit anti-mouse antibodies (CD9, flotillin, Hsp70; YAP1, bcl-2, cleaved-caspase 3, CTGF, TEAD1, ANP, and β-actin; Abcam; USA) at a dilution of 1:1,000. After overnight incubation at 4°C, the membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:10,000) for 1 h at room temperature. The immunobands were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime, PR China).
7
Immunoblot Analysis of Infected Fibroblasts
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Infected fibroblasts were collected and washed twice with pre-cold PBS and lysed in RIPA lysis buffer (Beyotime) containing 1× protease inhibitor (MCE) on ice for 30 min. Lysates were sonicated with Qsonica Q800R3 sonicator for 1 min at 40% amplitude with 5/7 s ON/OFF cycle followed by centrifugation at 15,000 rpm at 4 °C for 15 min. Protein concentrations in the cleared lysates were measured using BCA (Beyotime). Immunoblot analysis was performed by a simple western size-based protein assay (Protein Simple) following manufactures instructions. In brief, after loading of 750–1000 ng total protein onto each capillary, targeted protein level was identified using specific primary antibodies (E2F4, proteintech, 10923-1-AP; ATF3, aBCAm, ab207434; Tead1, aBCAm, ab133533; β-actin, abclonal, AC028; all 1:250 diluted). Chemiluminescence signals were analyzed using Compass software (Protein Simple).
8
Pancreatic Immunostaining and Morphometric Analysis
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Mouse pancreas were harvested, embedded in paraffin and sectioned to 5 µm thickness.
Immunostaining was performed as previously described (Lee et al., 2013) . Primary antibodies used were Glut2 (rabbit polyclonal, 1:200; Millipore), Insulin (guinea pig ployclonal, 1:500; Abcam), Glucagon (rabbit polyclonal, 1:200; Dako), Tead1 (rabbit polyclonal, 1:200; Abcam), Taz (rabbit polyclonal, 1:200; Santa Curz), Yap (rabbit poly clonal 1:200; Cell signaling), p16 (mouse monoclonal, 1:1,000; Abcam), Pdx1 (guinea pig polyclonal, 1:500; Abcam), Mafa (rabbit polyclonal, 1:100; Bethyl lab), Ucn3 (rabbit polyclonal, 1:2,000; MGI), Ki67 (rabbit poly clonalm 1:50; Abcam), Brdu (rat polyclonal, 1:1,000; Abcam), Caspase-3 (rabbit polyclonal, 1:200; Cell signaling), and Cleaved Caspase-3 (rabbit monoclonal, 1:200; Cell signaling). To measure total pancreatic islet area, pancreas paraffin blocks from mice were cut 5 µm thickness sections spaced 100 µm apart between each slide. 5 pancreatic sections per mouse were stained and measured islets area using ImageJ software. For assessing number of Brdu positive and Ki67 expression cells in β-cell, 3,000 to 5,000 cells were counted from mouse pancreatic sections in 3 to 4 mice from each group.
Immunostaining was performed as previously described (Lee et al., 2013) . Primary antibodies used were Glut2 (rabbit polyclonal, 1:200; Millipore), Insulin (guinea pig ployclonal, 1:500; Abcam), Glucagon (rabbit polyclonal, 1:200; Dako), Tead1 (rabbit polyclonal, 1:200; Abcam), Taz (rabbit polyclonal, 1:200; Santa Curz), Yap (rabbit poly clonal 1:200; Cell signaling), p16 (mouse monoclonal, 1:1,000; Abcam), Pdx1 (guinea pig polyclonal, 1:500; Abcam), Mafa (rabbit polyclonal, 1:100; Bethyl lab), Ucn3 (rabbit polyclonal, 1:2,000; MGI), Ki67 (rabbit poly clonalm 1:50; Abcam), Brdu (rat polyclonal, 1:1,000; Abcam), Caspase-3 (rabbit polyclonal, 1:200; Cell signaling), and Cleaved Caspase-3 (rabbit monoclonal, 1:200; Cell signaling). To measure total pancreatic islet area, pancreas paraffin blocks from mice were cut 5 µm thickness sections spaced 100 µm apart between each slide. 5 pancreatic sections per mouse were stained and measured islets area using ImageJ software. For assessing number of Brdu positive and Ki67 expression cells in β-cell, 3,000 to 5,000 cells were counted from mouse pancreatic sections in 3 to 4 mice from each group.
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