hNS1 cells cultivated in multiwell plates were dissociated with trypsin and centrifuged in PBS. The pellets were treated with lysis buffer (RIPA, Cell Signaling Technology, Danvers, MA, USA) to obtain cell extracts. Then, 50 μg of total proteins from cell extracts were loaded on sodium dodecyl sulphate-polyacrylamide gels (10%, BioRad, Hercules, CA, USA), electrophoresed (SDS-PAGE), and transferred to nitrocellulose membranes (GE Healthcare, Madrid, Spain). Membranes were blocked with milk (5%) and Tween20 (0.05%, Sigma) in TBS for 1 h and incubated overnight at 4 °C with mouse antibodies against APP (clone 22C11; 1:1000, Millipore, Merck, Darmstadt; Germany) and β-actin (1:1000, Sigma). Then, membranes were washed with Tween20 in TBS and incubated with HRP-conjugated antibody (horse anti-mouse peroxidase; 1:3000, Vector Laboratories, Newark, CA, USA) for 1 h. All antibodies used were diluted in milk and Tween20 in TBS. Visualization of immunoreactive bands was performed using ECL chemiluminescent system (Millipore) according to the manufacturer’s instruction. For quantification of blot images, densitometry analyses were realized using the software ImageJ v.1.54a. Band intensity was measured as a ratio of the protein of interest (APP using 22C11 antibody) to β-actin from three independent experiments.
Tween20
Manufactured by Vector Laboratories
Sourced in United States
Tween20 is a non-ionic detergent commonly used in biochemical applications. It is a polyoxyethylene sorbitan monolaurate that functions as a surfactant to solubilize and stabilize proteins and other biological molecules.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Radioimmunoprecipitation assay (ripa), by Cell Signaling Technology (2 mentions)
Ecl chemiluminescent system, by Merck Group (2 mentions)
Mouse antibodies against app clone 22c11, by Merck Group (2 mentions)
Sodium dodecyl sulphate polyacrylamide gels, by Bio-Rad (1 mentions)
Tween 20, by Thermo Fisher Scientific (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Vectashield plus dapi, by Vector Laboratories (1 mentions)
4 protocols using tween20
1
Western Blot Analysis of APP in hNS1 Cells
2
Immunofluorescence Staining of Rabbit Corneas
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Rabbit corneas from the organ culture system were embedded in ornithine carbamoyltransferase (OCT) (TissueTek; Sakura Finetek USA, Torrance, CA, USA), frozen using a dry ice/isopentane bath, and stored at −70°C until use. Cryosections (6 μm) on slides were fixed in −20°C methanol for 10 minutes and nonspecific binding blocked by incubation with 5% normal goat serum (NGS) (MilliporeSigma) in PBS with 0.05% Tween-20 (MilliporeSigma). Sections were incubated for 1 hour in 5 μg/mL antibody to human ADAM-17 (MAB 9304; R&D Systems, Minneapolis, MN, USA) in PBS with 1.5% NGS at room temperature, washed three times with PBS with 0.05% Tween-20, and incubated for 1 hour with goat anti-mouse immunoglobulin G (IgG)–Alexa Fluor 488 conjugate (Invitrogen) 1:1000 in PBS with 1.5% NGS. After washing three times with PBS with 0.05% Tween-20, sections were coverslipped using Vectasheild hard set antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).17 (link),18 (link)
3
Western Blot for β-Catenin and APP
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hNS1 cells cultivated in multiwell plates (at 4 days after nucleofection) were dissociated with trypsin and centrifuged in PBS. The pellets were treated with lysis buffer (RIPA, Cell Signaling) to obtain cell extracts. In all cases, 50 μg of total proteins from cell extracts were loaded on sodium dodecyl sulfate-polyacrylamide gels (10%, BioRad, Hercules, CA, USA), electrophoresed (SDS-PAGE), and transferred to nitrocellulose membranes (GE Healthcare). The membranes were blocked with milk (5%) and Tween20 (0.05%, Sigma) in TBS for 1 h and incubated overnight at 4 °C with rabbit antibodies against β-Catenin (1:1000, Cell Signaling) or mouse antibodies against APP (clone 22C11; 1:1000, Millipore) and β-Actin (1:1000, Sigma). Then, the membranes were washed with Tween20 in TBS and incubated with HRP-conjugated antibody (goat anti-rabbit peroxidase; 1:3000, Vector Laboratories, Newark, CA, USA, or horse anti-mouse peroxidase; 1:3000, Vector Laboratories) for 1 h. All the antibodies were diluted in milk and Tween20 in TBS. Visualization of immunoreactive bands was performed using an ECL chemiluminescent system (Millipore) according to the manufacturer’s instructions.
4
Immunocytochemical Analysis of Osteogenic Markers
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At the appropriate time points samples were removed from culture, washed with PBS and fixed with 4% paraformaldehyde/2% sucrose solution. Samples were stained for von Kossa using the previously described methods and prepared for immunocytochemical analysis of osteocalcin (10µg/ml R&D Systems) /actin (Oregon Green Phallodin, 5µg/ml, Invitrogen UK)
or CBFA1 (5µg/ml R&D Systems)/actin using the following methods. Following fixation samples were washed and permeabilised with a 0.05% Triton x100 solution in PBS for 5 minutes at 4 o C then incubated with the primary antibody, diluted in 1%BSA: PBS, overnight at 4 o C. Samples were then washed with a 0.1% Tween 20 (ICN Biomedicals) followed by incubation with Rhodamine conjugated goat IgG fraction to mouse IgG (Invitrogen) 1µg/ml in 1%BSA:PBS for osteocalcin and for CBFA1 primary antibodies a Texas Red goat anti-rat secondary, 1µg/ml in 1%BSA:PBS (Invitrogen) was used. Samples were incubated with the secondary antibodies for 1 hour at 37 o C, followed by washing with the Tween 20 solution and incubation with Oregon Green Phallodin, diluted in PBS, for 30 minutes at 4 o C.
Samples were washed then mounted onto microscope slides using Vectashield plus DAPI (Vector, UK) and visualised using confocal microscopy, Zeiss LSM510.
or CBFA1 (5µg/ml R&D Systems)/actin using the following methods. Following fixation samples were washed and permeabilised with a 0.05% Triton x100 solution in PBS for 5 minutes at 4 o C then incubated with the primary antibody, diluted in 1%BSA: PBS, overnight at 4 o C. Samples were then washed with a 0.1% Tween 20 (ICN Biomedicals) followed by incubation with Rhodamine conjugated goat IgG fraction to mouse IgG (Invitrogen) 1µg/ml in 1%BSA:PBS for osteocalcin and for CBFA1 primary antibodies a Texas Red goat anti-rat secondary, 1µg/ml in 1%BSA:PBS (Invitrogen) was used. Samples were incubated with the secondary antibodies for 1 hour at 37 o C, followed by washing with the Tween 20 solution and incubation with Oregon Green Phallodin, diluted in PBS, for 30 minutes at 4 o C.
Samples were washed then mounted onto microscope slides using Vectashield plus DAPI (Vector, UK) and visualised using confocal microscopy, Zeiss LSM510.
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