Mice corneas (n = 6/group/time) were harvested at 3 days p.i. Protein extraction and concentration determination were performed as described previously.4 (link) The protein was separated on 12% acrylamide SDS-PAGE and transferred to polyvinylidene difluoride (PVDF; Solarbio) membrane. Membranes were washed in PBS containing 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) (PBST) three times. After being blocked with blocking buffer (Beyotime, Jiangsu, China) at 37°C for 2 hours, the membrane was incubated with antibodies to GAPDH (1:1000; Elabscience, Wuhan, China), CXCL-1 (1:1000; Affinity Biosciences, Jiangsu, China), LC3B (1:1000; Cell Signaling Technology, Danvers, MA, USA), SQSTM1/p62 (1:1000; Cell Signaling Technology), Beclin-1 (1:1000; Cell Signaling Technology), LAMP-1 (1:1000; Cell Signaling Technology), IL-1β (1:1000; NOVUS, Littleton, CO, USA), IL-18 (1:500; Abcam, Cambridge, MA, USA), HMGB1 (1:1000; Abcam), TNF-α (1:2000; Proteintech Group, Wuhan, China), or IL-10 (1:1000; Abcam) at 4°C overnight. Membranes were washed in PBS containing 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) (PBST) three times. Membranes were incubated with corresponding peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37°C for 1 hour. Membranes were developed by chemiluminescence (ECL; Thermo Fisher Scientific, Waltham, MA, USA).
Tween 20
Manufactured by Bio-Rad
Sourced in United States, Switzerland, Canada
Tween-20 is a non-ionic detergent commonly used in biochemical applications. It is a polyoxyethylene sorbitan monolaurate, designed to facilitate the solubilization and stabilization of proteins and other biological molecules. Tween-20 is widely used in various laboratory techniques, such as Western blotting, ELISA, and immunoprecipitation, to help maintain the integrity and activity of the target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (20 mentions)
Triton x 100, by Merck Group (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Ammonium persulfate, by Bio-Rad (8 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (7 mentions)
Non fat dry milk, by Bio-Rad (7 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Acrylamide, by Bio-Rad (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (6 mentions)
Triton x 100, by Bio-Rad (6 mentions)
Ethanol, by Merck Group (6 mentions)
Laemmli sample buffer, by Bio-Rad (5 mentions)
Goat serum, by Thermo Fisher Scientific (5 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (5 mentions)
Enhanced chemiluminescence, by GE Healthcare (4 mentions)
Tween 20, by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Corning (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Sucrose, by Merck Group (4 mentions)
140 protocols using tween 20
1
Western Blot Analysis of Corneal Proteins
2
SARS-CoV-2 Peptide Pool Stimulation and Flow Cytometry
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PBMCs were cultured in a 96-well plate and stimulated, as previously mentioned in the T-cells stimulation and treatment section, with an extra step involving the addition of 1 μL/200μL Monensin GolgiPlug (eBioscience, Waltham, MA, USA) after 6 h of SARS-CoV-2 peptide pool stimulation. During post-incubation, cells were washed, and the cell surface was stained with anti-CD4-PerCP5.5 (clone RM4-5; Invitrogen) and anti-CD8a-APC-eFluor 780 (clone RPA-T8; Invitrogen, Waltham, MA, USA) for 1 h on ice before being fixed with 1% paraformaldehyde and kept overnight at 4 °C. After permeabilization with 0.1% Tween 20 (Bio rad, Hercules, CA, USA) at room temperature for 20 min, the cells were intracellularly stained with TNF-α-PE (Beckman Coulter, Brea, CA, USA), IFN-γ Monoclonal Ab CC302 (Invitrogen, Waltham, MA, USA), and IFN-γ-Alexa Fluor 488 goat anti-mouse IgG1 (Invitrogen, Waltham, MA, USA) for 1 h on ice. Thereafter, the cells were washed and acquired using a BD FACS Canto II Flow Cytometer (BD Biosciences, San Jose, CA, USA). BD FACSDiva software was used to analyze the results. The gating strategy (Figure S4 ) was performed by BD LSRFortessa.
3
Western Blot Analysis of Liver Proteins
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Liver samples were lysed with ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate dibasic and 1% Nonidet P-40) in the presence of protease and phosphatase inhibitor mixtures (Thermo Fisher Scientific), 1 mM DTT and 2 mM Na3VO4. Total sample lysates were mixed with 6× SDS loading buffer (Alfa-Aesar) and boiled for 5 min. Protein samples (10–100 μg) were loaded and separated on 4–20% gradient SDS/PAGE gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% BSA in 1× TBS supplemented with 0.1% Tween 20 (Bio-Rad) and incubated with the following antibodies: GS (610518, BD), GLS2 (150474, Abcamab), and GAPDH (14C10, Cell Signaling). Bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:10,000; Jackson ImmunoResearch) and enhanced chemiluminescence reagent (Thermo Fisher Scientific). Band intensities were quantified in ImageJ software.
4
Quantification of PPARγ Protein in Mouse Hippocampus
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Western blotting was performed as previously described [38] with modifications. Protein samples from the HP of all eight groups of mice (Table 2) were used. Proteins (15µg) were separated on 4-20% Bolt Bis-Tris Plus gels (Invitrogen), as described previously [15, 25] . The gel was subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and blocked with 5% (w/v) non-fat milk tris-buffered saline (TBS) with 0.1% Tween-20 (Bio-Rad) for 1 hour at room temperature. The membrane was then incubated with rabbit anti-PPARγ (1:1000, Cell Signaling) primary antibody at 4°C overnight. Subsequently, a corresponding anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; DAKO) was applied. Binding was visualized with enhanced chemiluminescence solution using Luminata Forte Western horseradish peroxidase (HRP) substrate (Millipore). Band intensity was measured as the sum optical density by using ImageJ software (version1.4; NIH) and normalized to control labelling of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000, Millipore).
5
Immunofluorescence Staining of RPE Cells
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RPE cells were cultured in 4-chambered culture slides (Falcon, Thermo Fisher). The cells were rinsed in phosphate-buffered saline (PBS) for 3 min, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Tween 20 (Bio-Rad) in PBS. After 30-min blocking with 5% normal goat serum, the cells were incubated with the primary antibodies (Supplementary Table 1) overnight at 4 • C and then incubated with the corresponding secondary antibodies for 30 min at room temperature. All antibodies were diluted in PBS. The slides were mounted with mounting medium containing DAPI (Vector Laboratories) and examined with a KEYENCE fluorescence microscope (BZ-X700, San Diego, CA, USA).
6
Immunofluorescence Analysis of Cellular Signaling
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Bovine serum albumin (BSA, A7906, Sigma), 16% Formaldehyde (w/v), methanol-free (Cat #28908, Pierce™ ThermoFisher Scientific), DAPI (D1306, ThermoFisher Scientific), Hydrogen peroxide solution (H2O2, 323381, Sigma), Stattic (S7947, Sigma), U0126 (9930, CST), Triton X-100 (T8787, Sigma), Tween-20 (170-6531, Bio-Rad).
7
Immunoblotting of Cellular Proteins
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Cells were collected in a sodium dodecyl sulfate (SDS) lysis buffer. After sonication, the proteins were subjected to electrophoresis in a 5–20% SDS–polyacrylamide gel, and then transferred onto a polyvinylidene difluoride membrane (ATTO), followed by overnight blocking with 5% skimmed milk in phosphate-buffered saline with Tween 20 (Bio-Rad, Hercules, CA, USA). The membrane was probed with antibodies specific to MAP2K3 (Cell Signaling Technology, Boston, MA, USA), GAPDH (Santa Cruz), and/or β-tubulin (Abcam K.K., Tokyo, Japan). After washing the membrane, it was incubated with secondary horseradish peroxidase-conjugated antibodies for an hour. The signals were detected with enhanced chemiluminescence (GE Healthcare, Tokyo, Japan) and scanned with an image analyzer LAS-4000 mini (Fuji Film, Tokyo, Japan), as previously described [13 (link)].
8
Evaluation of 7,8-DHF Effects on Melanoma Cells
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SK-MEL-2 and G-361 cells were treated with various concentrations of 7,8-DHF for different incubation times (100, 200, and 300 µM, for 12 and 24 h) and washed twice with DPBS. Cells were homogenized well with a protein extraction solution (RIPA) (ELPIS Biotech Inc., Daejeon, Korea). The extracted proteins were quantified using the Pierce® BCA Protein Assay kit (Thermo Fisher Scientific lnc., Waltham, MA, USA). Equal amounts of protein samples were separated by 8%, 10%, and 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) along with protein markers and then transferred to polyvinylidene difluoride (PVDF) blotting membranes. The membranes were blocked at room temperature for one hour with 5% non-fat dried milk in 1 × TBS (Biosesang, Seongnam, Gyeonggi, Korea) containing 0.1% Tween-20 (Bio-Rad Inc., CA, USA) and then incubated overnight at 4 ℃ with specific primary antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for two hours, immunoreactive bands were visualized using a SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific lnc., Waltham, MA, USA).
9
In Vitro Insulin Signaling Assay
10
Particle Internalization in N27 Cells
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