The cells were harvested with radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio) containing phenylmethanesulfonyl fluoride (PMSF; Solarbio) and protease inhibitor cocktail (Solarbio). The lysates were removed through centrifugation at 12,000 × g for 15 min at 4°C. The cell protein lysates were quantified, separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and then blocked with 5% skim milk (BD Biosciences) prepared in 1× TBST with 0.05% Tween-20 (Solarbio). Subsequently, the membrane was incubated with specific primary antibodies, washed, and probed with horseradish-peroxidase-conjugated secondary antibody. Several antibodies such as E-cadherin (1:2,000 dilution, rabbit anti-human polyclonal antibody, #20874-1-AP; Proteintech, Wuhan, China), N-cadherin (1:6,000 dilution, rabbit anti-human polyclonal antibody, #22018-1-AP; Proteintech), Vimentin (1:2,000 dilution, rabbit anti-human polyclonal antibody, #10366-1-AP; Proteintech), and GAPDH (1:6,000 dilution, rabbit anti-human polyclonal antibody, #10494-1-AP; Proteintech) were used. Furthermore, the signals were detected using Molecular Imager® (Tanon, Shanghai, China).
Tween 20
Manufactured by Solarbio
Sourced in China, United States
Tween-20 is a nonionic surfactant commonly used in biological research applications. It is a polyoxyethylene sorbitan monolaureate with a hydrophilic-lipophilic balance (HLB) value of 16.7. Tween-20 is soluble in water and is used to facilitate the solubilization and dispersion of compounds in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Triton x 100, by Solarbio (6 mentions)
Ripa lysis buffer, by Solarbio (4 mentions)
Bovine serum albumin (bsa), by Solarbio (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (3 mentions)
Tris buffered saline (tbs), by Solarbio (3 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Reserpine, by Maclin Biochemical Technology (3 mentions)
Eugenol, by Maclin Biochemical Technology (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Xyz3060 dispenser, by BioDot (2 mentions)
N hydroxysuccinimide nhs, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Phosphate buffered saline (pbs), by Solarbio (2 mentions)
Bca kit, by Beyotime (2 mentions)
Sodium chloride (nacl), by Sinopharm (2 mentions)
Phosphate buffer solution (pbs), by Solarbio (2 mentions)
55 protocols using tween 20
1
Western Blot Analysis of EMT Markers
2
Colocalization of CD71 and HFn-FITC in C666-1 Cells
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Fluorescence microscopy analysis
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).
3
In Situ Hybridization for Viral RNA Detection
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ISH was performed according to the previously published procedure (47 (link), 48 ), with some modifications. After dewaxing and rehydration, tissue sections were treated with HCl (0.2 mol/liter; 20 min) and proteinase K (20 μg/ml; 30 min; 37°C) (TaKaRa). After washing with phosphate buffer containing Tween 20 (PBST), the slides were prehybridized for 4 h at 42°C in the mixture of 50% formamide, 5× saline citrate (SSC) (Solarbio), 0.1% Tween 20 (Solarbio), 1.9 g/liter citric acid monohydrate, 500 μg/ml tRNA (Sigma), and 50 μg/ml heparin sodium (Solarbio). Hybridization was performed in the same solution mixed with 1 mg/ml DIG-labeled RNA probe at 42°C for 16 h. To detect probes hybridized with viral RNA, we incubated tissue sections with anti-DIG-AP Fab fragments (Roche) for 12 h at 4°C and then stained the hybridization with BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (4-nitroblue tetrazolium chloride) (Roche). The slides were counterstained with Bismarck brown. Healthy prawns served as a negative control by performing the same protocol.
4
Optimized Cell Culture Protocol for PAX7 Analysis
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The following materials are used in the cell culture: L-leu (#L8912), lysine (Lys, #L9037), arginine (Arg, #A6969), Collagen Ⅰ (#SCR103), Basic Fibroblast Growth Factor (bFGF, #13256-029), BSA (A1933), and DMSO (#C6295), which were purchased from Sigma-Aldrich, USA. DMEM (#11995065), L-leu-deprived DMEM powder (#88425), fetal bovine serum (FBS, #10099141C), Antibiotics-antimycotic (#15240062), and Dulbecco phosphate-buffered saline (DPBS, #14190144) were purchased from Gibco, Life, Technologies, Grand Island, NY, USA. Culture dish (10 and 6 cm), Cell ware 6, 24, and 96-well plates, Matrigel and cell strainer, were purchased from Corning, NY, USA. Accutase, Cell Detachment Solution was purchased from innovative cell technologies, SD, USA. Mouse monoclonal anti-PAX7 (#sc-81648) was purchased from Santa Cruz Biotechnology, CA, USA. Donkey anti-Mouse IgG (H + L) was purchased from Invitrogen (#A10036, Invitrogen, Carlsbad, CA, USA). DAPI (#C0065), Triton X-100 (#9002-93-1), and Tween 20 (#9008-64-5) were purchased from Solarbio (Beijing, China). CCK 8 was purchased from Good Laboratory Practice Bioscience, Montclair, CA, USA.
5
Immunofluorescence Staining of BHK-21 Cells
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BHK-21 cells were seeded in 12-well plates, and after 24 h of culture, they were rinsed with phosphate-buffered saline (PBS) (Solarbio, Beijing, China) and fixed using methanol (Solarbio) for 10 min at room temperature. The cells were washed three times with PBS and then incubated in blocking buffer [3% bovine serum albumin (BSA) (BOSTER, Beijing, China) in Tris-buffered saline (Solarbio) and Tween20 (Solarbio)] for 1 h at room temperature. The cells were then incubated with an anti-E monoclonal antibody (Abcam, Cambridge, UK) (1:20) in PBS with 3% BSA overnight at 4°C. After the cells were washed three times in PBS, they were incubated with a fluorescently labeled secondary antibody—fluorescein isothiocyanate-conjugated goat antimouse antibody (ZSGB-Bio, Beijing, China)—in the dark at 37°C for 1 h. The nuclei were washed with PBS three times and stained with 4,6-diamidino-2-phenylindole (DAPI) (Solarbio). Slides were imaged under a fluorescence microscope (Olympus, Tokyo, Japan).
6
Rice Blast Resistance Screening Protocol
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The Magnaporthe oryzae Hoku1 provided by the Institute of Plant Protection of Chinese Academy of Agricultural Sciences was used for inoculation. For the panicle blast resistance identification, a 2 mL spore suspension with a concentration of 1 × 105/mL was used for injection at the booting stage of rice. A total of 9 panicles of each line were injected with 3 strains. The percentage of diseased grains was calculated after three weeks. Grains infected with spore-forming areas were defined as diseased grains [40 (link)].
For the leaf blast resistance identification, a spore suspension with a concentration of 1 × 105/mL was used, which was combined with 0.02% Tween 20 (Solarbio, Beijing, China, Cat#T8220) [68 (link),69 (link)]. According to the method of Zhu et al. (2012) [68 (link)], each seedling tray is inoculated with 60 mL of spore suspension by spraying the seedlings with a spray gun connected to an air pump. After inoculation, seedling trays were placed in a dark environment at 95–100% relative humidity and 27 °C for 24 h and then transferred to a greenhouse with the temperature maintained at 27 °C. The length and number of lesions were recorded after six days. Six plants for each line and three replicates were measured, and the average was calculated for the resistance phenotype.
For the leaf blast resistance identification, a spore suspension with a concentration of 1 × 105/mL was used, which was combined with 0.02% Tween 20 (Solarbio, Beijing, China, Cat#T8220) [68 (link),69 (link)]. According to the method of Zhu et al. (2012) [68 (link)], each seedling tray is inoculated with 60 mL of spore suspension by spraying the seedlings with a spray gun connected to an air pump. After inoculation, seedling trays were placed in a dark environment at 95–100% relative humidity and 27 °C for 24 h and then transferred to a greenhouse with the temperature maintained at 27 °C. The length and number of lesions were recorded after six days. Six plants for each line and three replicates were measured, and the average was calculated for the resistance phenotype.
7
Western Blot Analysis of RUNX-2 Expression
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Following treatment, the protein extracts of the cells were harvested in RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and protein concentration in the cell lysates was quantified using the BCA Protein Assay kit. Proteins (15 µg) were then separated by SDS-PAGE on 10% gels and transferred to nitrocellulose membranes, which were blocked with 2.5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) in Tris-buffered saline with 0.1% Tween-20 (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h. Membranes were then incubated with the following primary antibodies at 4˚C for 15 h: Anti-RUNX-2 (1:200; cat. no. sc-390351) and anti-β-actin (1:1,000; cat. no. sc-8432) (both from Santa Cruz Biotechnology, Inc.). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at 37˚C for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence detection reagent (Pierce; Thermo Fisher Scientific, Inc.). β-actin in the cell lysates was used as a loading control and data were normalized against the corresponding optical density of β-actin. ImageJ 1.48 software (National Institutes of Health) was used for semi-quantification of bands.
8
Aβ25-35-Induced Apoptosis Pathway Analysis
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Aβ25-35 was purchased from Sigma-Aldrich (A4559, St Louis, MO, USA). Donepezil Hydrochloride Tablet (Don) was purchased from Eisai, Shanghai, China. HPTQC (Z20080006) was provided by the First Affiliated Hospital of Anhui University of Chinese Medicine. Annexin V-PI kit (Lot.600125a) was purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Protein lysate (Lot. 70126373), PMSF (Lot. 329-98-6), Tween-20 (Lot. 20CS180) and Tris (Lot. 1130K07) were purchased from Beijing Solarbio Science & Technology Co., Ltd. The following antibodies, Caspase3 (Lot. 89Z0306), Caspase9 (Lot. 74z1125), Caspase12 (Lot. 76r9498), CHOP (Lot. 37s3359), and GRP78 (Lot.64f2874), were from Affinity Biosciences (Cincinnati, OH, USA). PVDF membrane (Lot. RB88250) was purchased from Millipore. TRIzol kit (Lot. 15596-026) was purchased from Ambion (Carlsbad, CA, USA). FBS (Lot.10099140C) was purchased from Bio-Rad Laboratories.
9
Genistein Protocol for Cell Studies
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Genistein dry powder was purchased from Med-ChemExpress (MCE, USA), a Roswell Park Memorial Institute (RPMI)-1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Biological Industries (Israel), fetal bovine serum (FBS) was purchased from Gibco (USA), and small interfering ribonucleic acid (siRNA) was acquired from GenePharma (Shanghai, China). The inhibitor (ASP-9521) was purchased from MCE (USA), while the streptavidin-biotin complex (SABC) kit was purchased from Solarbio (Beijing, China), and the ki-67 cell proliferation kit was purchased from Gibco (USA). Western and immunoprecipitated cell lysates were purchased from Biyuntian (Beijing, China); the bicinchoninic acid (BCA) protein quantification kit and Tween-20 were purchased from Solarbio (Beijing, China); the PSA/AKR1C3 monoclonal antibody was purchased from Abcam (USA); beta (β)-ACTIN monoclonal, rabbit secondary, and mouse secondary antibodies were purchased from Zhongshan Jinqiao (Beijing, China); a western chemiluminescence horseradish peroxidase substrate was purchased from Millipore (USA); and a 4x loading buffer was purchased from Solarbio (Beijing, China).
10
Magnetic Bead-Based Sandwich ELISA for CEA and VEGF
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Human VEGF165 was purchased from Peprotech Corporation (USA). CEA was obtained from Abcam Corporation (USA). Human CEA ELISA kit was acquired from Huaketai Biotechnology Co. Ltd. (Beijing, China). Human VEGF165 ELISA kit was from Miblo Co. Ltd. (Shanghai, China). Alkaline phosphatase-labeled streptavidin (ALP-SA) was purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Tween-20, Biotin, Dimethyl sulfoxide (DMSO) and Tris (hydroxymethyl) aminomethane-hydrochloric acid (Tris-HCl) were obtained from Solarbio Co. Ltd. (Beijing, China). HCl, NaCl, KCl, and NaOH were acquired from Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). Streptavidin modified magnetic beads (MBs, 300 nm) were from Biomag Biotechnology Co. Ltd. (Wuxi, China). Luminol was acquired from Sigma corporation (USA). 3-(2-Spiroadamatane)-4-methoxy-4-(3-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) was from Huaxia reagent Co. Ltd. (Chengdu, China). 4-Phenylphenol (BIP) was purchased from Aladdin Co. Ltd. (Shanghai, China). Ultrapure water (18.3 MU cm) was obtained from a Milli-Q system (Waters, Milford, MA, USA) and used for all experiments.
All oligonucleotides employed in the work were synthesized and purified by Sangon Biotechnology Corporation (Shanghai, China) and the sequences are listed inTable 1 .
All oligonucleotides employed in the work were synthesized and purified by Sangon Biotechnology Corporation (Shanghai, China) and the sequences are listed in
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