Cells were cultured in polymer dishes as described previously5 (link). Cells were fixed for 20 min using 4% (by weight) formaldehyde solution (Sigma-Aldrich 252,549). Subsequently, cells were permeabilized with 0.5% by volume Tween 20 (Sigma-Aldrich P1379) in PBS for 20 min, and then blocked with 1% by weight bovine serum albumin (BSA, Sigma-Aldrich A7906), 22.5 mg/mL glycine in PBST (0.1% by volume Tween 20 in PBS) for 30 min. Cells were incubated with the primary antibodies diluted in 1% BSA in PBST at 4 °C overnight. After that, cells were incubated with the secondary antibodies in 1% BSA at room temperature for 1 h. Finally, the nuclei were stained with DAPI or Hoechst (Thermofisher Scientific R37606 or R37605) fluorescent dyes. The primary antibody used: Atf2 (abcam ab32019), Beta-Catenin (abcam ab19381), GEF-H1 (abcam ab155785), Lef1 (abcam ab137872), Oct4 (abcam ab19857), RNA pol II (abcam ab5408), Smad4 (CST 46,535).
Ab19381
Manufactured by Abcam
Sourced in United Kingdom
Ab19381 is a primary antibody that can be used for the detection of a specific protein target in various experimental applications. The product information does not provide additional details about its core function or intended use.
Automatically generated - may contain errors
Lab products found in correlation
A7906, by Merck Group (1 mentions)
R37606, by Thermo Fisher Scientific (1 mentions)
Ab19857, by Abcam (1 mentions)
R37605, by Thermo Fisher Scientific (1 mentions)
P1379, by Merck Group (1 mentions)
Ab137872, by Abcam (1 mentions)
Ab155785, by Abcam (1 mentions)
Ab32019, by Abcam (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ab131097, by Abcam (1 mentions)
Ab93926, by Abcam (1 mentions)
Ab151724, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab15251, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Ab109367, by Abcam (1 mentions)
Ab96899, by Abcam (1 mentions)
Goat anti mouse dylight 647, by Abcam (1 mentions)
4 protocols using ab19381
1
Immunofluorescence Staining of Cells
2
Western Blot Analysis of Signaling Proteins
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The western blot assay was conducted as previously reported (25 (link)). Total protein was isolated from U251 and A172 cell lines with RIPA lysis buffer (Invitrogen) and quantified using the bicinchoninic acid (BCA) method. The proteins were separated by 10% SDS-PAGE. Equal amount of protein was transferred to a PVDF membrane and incubated at 4°C for 24 h with anti-SLIT1 (1:5,000, ab151724, Abcam, United Kingdom), anti-Wnt (1:1,000, ab15251, Abcam), anti-β-catenin (1:5,000, ab19381 Abcam), anti-p-Gsk-3β (1:1,000, ab131097, Abcam), anti-Gsk-3β (1:1,000, ab93926, Abcam), or anti-GAPDH (1:2000, ab8245, Abcam). The membrane was incubated with HRP-conjugated IgG secondary antibody (1:10,000, ab97051, Abcam) at room temperature for 2 h. The protein blots were visualized using the ECL method. GAPDH served as an internal control.
3
Immunostaining of β-Catenin in Cultured Cells
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Cells were cultured in an eight-well chamber slide (154534; Thermo Fisher Scientific) to confluence, four wells per cell type. The cells were then fixed using 4% PFA for 20 min at RT and blocked using 1% BSA. Immunofluorescent staining was performed using the primary antibody of β-catenin (ab19381, 1:100; Abcam) overnight at 4°C, and the secondary antibody of anti-mouse Alexa Fluor 647 (A-21240, 1:400; Thermo Fisher Scientific) for 1 h at RT. Nuclei were counterstained using DAPI. The cells were then imaged using a Zeiss LSM 880 confocal microscope with a 20x objective.
4
Immunofluorescence Staining of PRDX2 and β-Catenin
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Coverslips were plated in 6-well plates, and HepG2 cells were added onto them. The following day, cells were fixed with 4% paraformaldehyde, then permeabilized by treating them with PBS containing 0.1% Triton X-100 for 15 min. The slides were washed three times with PBS, then blocked with 2% BSA for 30–45 min at room temperature, The slides were incubated overnight with the following primary antibodies: anti-PRDX2 rabbit monoclonal antibody (1:250, ab109367, Abcam) and anti-β-catenin mouse monoclonal antibody (1:200, ab19381, Abcam) at 4 degrees. The next day, the slides were washed three times with PBST for 5 min each time. Then, the slides were incubated with fluorescent secondary antibody: Goat anti-rabbit-DyLight® 488 (1:1000, ab96899, Abcam) and Goat anti-mouse-DyLight® 647 (1:1000, ab150115, Abcam) for one hour at room temperature in the dark. Afterwards, the slides were washed three times with PBST and observed using a fluorescent microscope.
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