The treated myocardiocytes were cultured in 6-well plates for 12 h at 37°C, washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and then incubated with 0.1% Triton X-100 and 1% BSA for 30 min at room temperature. Myocardiocytes were incubated with primary anti-mouse antibodies against Bim (1:1,000), CHOP (1:1,000), activating transcription factor 4 (ATF4; 1:1,000; product code ab216839; Abcam), inositol-requiring enzyme 1α (IRE1α; 1:1,000; product code ab37073; Abcam), thiobarbituric acid reactive substances (TBARS; 1:1,000; ab118970; Abcam), reactive oxygen species (ROS; 1:1,000; ab186027; Abcam), H2O2 (1:1,000; ab138874; Abcam) overnight at 4°C. Myocardiocytes were washed with PBS and incubated with corresponding anti-rabbit secondary antibody (1:2,000, product code ab7090; Abcam) for 2 h at 25°C. Subsequently, myocardiocytes were stained with 5% DAPI for 30 min at room temperature. Images of myocardiocytes were captured using a Zeiss confocal spectral microscope (Carl Zeiss AG) at a magnification of ×100.
Ab138874
Manufactured by Abcam
Sourced in United States
Ab138874 is a recombinant monoclonal antibody that recognizes the Glutathione S-Transferase (GST) tag. The antibody is produced in HEK293 cells and is purified by affinity chromatography. It can be used for the detection and purification of GST-tagged proteins.
Automatically generated - may contain errors
2 protocols using ab138874
1
Immunofluorescence Analysis of Myocardial Cells
2
Quantifying Superoxide and Hydrogen Peroxide
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Superoxide production was determined by staining CB-ECFCs with dihydroethidium (DHE; Sigma, St. Louis, MO, USA), which is specifically oxidised to ethidium by superoxide, while hydrogen peroxide production was quantified using a commercially available assay kit (ab138874, Abcam), according to the manufacturer’s instructions. For superoxide assay, cells were seeded on a 24-well plate at a density of 2 × 104 per well. For hydrogen peroxide assay, the cells were plated on a 96-well plate at a density of 5 × 103 per well, followed by culture under either hypoxia or normoxia for 48 h. Cells were then washed twice with a PBS and incubated with 10 µmol/L DHE in HBSS or AbGreen hydrogen peroxide indicator solution for 1 h at 37 °C. The staining solutions were replaced with HBSS for fluorescent imaging (Leica DMi8) with quantification in three wells analysing three images/well using LAS X v3.7.4 (Leica).
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