After the experimental treatments of each group, the HEI‐OC1 cells and cochlear basement membranes were fixed with 4% paraformaldehyde (PFA) for 30 min and were punched with 0.1% Triton X‐100 and 1% Triton X‐100 for 30 min, respectively. Then, the samples were treated with PBT‐1 blocking solution for 1 h, and incubated with PBT‐1 diluted primary antibody in a refrigerator (4°C) overnight. The primary antibody dilution was aspirated off and the samples were then incubated with PBT‐2 diluted secondary antibody and DAPI at 37°C for 1 h. The sample was sealed, observed and imaged under a fluorescent inverted microscope (Leica Microsystems, Biberach, Germany). The antibodies used included Myosin 7a (1:500, 138–1, DSHB, USA), NRF2 (1:200, A1244, ABclonal, China), HO‐1 (1:200, A1346, ABclonal, China) and GPX4 (1:200, AB125066, Abcam, UK).
A1244
Manufactured by ABclonal
Sourced in China
The A1244 is a laboratory equipment designed for general use in scientific research and experiments. It serves as a primary tool for various applications that require precise measurement and analysis. The core function of the A1244 is to provide consistent and reliable performance to support the needs of researchers and scientists in their work.
Automatically generated - may contain errors
Lab products found in correlation
A1346, by ABclonal (2 mentions)
Ab125066, by Abcam (2 mentions)
Inverted fluorescent microscope, by Leica (1 mentions)
A19586, by ABclonal (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab175186, by Abcam (1 mentions)
Gb11001, by Wuhan Servicebio Technology (1 mentions)
A0216, by Beyotime (1 mentions)
A5865, by ABclonal (1 mentions)
A19544, by ABclonal (1 mentions)
A0208, by Beyotime (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Ab70699, by Abcam (1 mentions)
Las 4000, by Cytiva (1 mentions)
A13685, by ABclonal (1 mentions)
A11243, by ABclonal (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence assay, by Thermo Fisher Scientific (1 mentions)
Anti ha 3724, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using a1244
1
Immunofluorescence Analysis of Cochlear Cells
2
Protein Extraction and Western Blot Analysis
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Each experimental group was washed twice with precooled PBS at 4°C, and the whole proteins were extracted by adding lysis solution RIPA (P0013, Beyotim, China) containing both phosphatase and protease inhibitors, lysed for 30 min at 4°C, vortexed every 5 min, and then centrifuged at 12,000g for 20 min at 4°C. The protein contents were measured with the BCA kit (P0010, Beyotime, China), added to the loading buffer, and then denatured by heating. After the protein samples were electrophoresed by 10% and 12% SDS‐PAGE, the proteins were transferred to PVDF membranes by wet transfer method. After blocking for 1 h, primary antibodies, including NRF2 (A1244, ABclonal, China), HO‐1 (A1346, ABclonal, China), NQO1 (A19586, ABclonal, China), GPX4 (ab125066, Abcam, UK), SLC7A11 (ab175186, Abcam, UK) and β‐actin (GB11001, Servicebio, China), all diluted 1:1000, were added and incubated overnight at 4°C. Then, the corresponding secondary antibody dilutions (1:5000) were added and incubated for 1 h. The target protein was analysed semiquantitatively using a fully automated chemiluminescence image analyser and ImageJ software for grayscale analysis of protein bands, with β‐actin as an internal reference.
3
Western Blot Analysis of Cellular Proteins
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Fresh tissue and cells were disrupted with a high-strength RIPA lysis buffer, followed by 30 min of standing and centrifugation at 12,000 rpm at 4°C for 10 min. The protein content of the supernatant was quantified using a bicinchoninic acid (BCA) standard curve. Ten microliters of samples with equal amounts of total protein were added to 12.5% or 7.5% gels and electrophoresed at a stable voltage of 200 V. The gels were then transferred onto a PVDF membrane at a constant current of 400 mA. After blocking with 5% skimmed milk for 2 h, the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-Bax (1:2000, 50599-2-Ig, Proteintech), rabbit anti-Bcl-2 (1:2000, 26593-1-AP, Proteintech), mouse anti-GPX4 (1:2000, 67763-1-Ig, Proteintech), rabbit anti-Nrf2 (1:3000, A1244, ABclonal), rabbit anti-CD71 (1:1000, A5865, ABclonal), rabbit anti-FTH1 (1:1000, A19544, ABclonal), rabbit anti-DMT1 (1:2000, 20507-1-AP, Proteintech), rabbit anti-Fpn1 (1:1000, 26601-1-AP, Proteintech), and mouse anti-β-actin (1:1000, 66009-1-Ig, Proteintech). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:3000, A0208, Beyotime) and goat anti-mouse (1:1000, A0216, Beyotime) antibodies were added for 1 h, followed by enhanced chemiluminescence (ECL) visualization.
4
Western Blot Analysis of Liver Tissues
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Western blot analysis of the liver tissues was performed according to the method described by Wu [31 (link)]. The primary antibodies were anti-Nrf2 (A1244, ABclonal, Wuhan, China), anti-Keap1 (A1820, ABclonal, Wuhan, China), and anti-GAPDH (ab70699, Abcam, Cambridge, UK). The bands were acquired using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Wuxi, China) and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Protein Signaling
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Tissue protein was prepared in lysis buffer with a protease and phosphatase inhibitors cocktail (TargetMol, Wellesley Hills, MA, USA). The total protein content was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. The proteins were denatured and resolved using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were then blocked with 5% non-fat milk for 2 h at room temperature and incubated with specific primary antibodies at 4 °C overnight. Following incubation with horseradish peroxidase–conjugated secondary antibodies (1:2000; Cell Signaling Technology) for 2 h at room temperature, and the bands were detected using an enhanced chemiluminescence assay (Thermo Fisher Scientific). The primary antibodies used for western blotting were anti-HA（#3724, 1:1000; Cell Signaling Technology), anti-Nrf2 (A1244, 1:1000; Abclonal Technology), anti-p-Nrf2 (AP1133, 1:1000; Abclonal Technology), anti-SLC7A11 (A13685, 1:1000; Abclonal Technology), anti-MLKL (A21894, 1:1000; Abclonal Technology), anti-p-MLKL (AP1173, 1:1000; Abclonal Technology), anti-GPX4 (A11243, 1:1000; Abclonal Technology), anti-GAPDH (10494-1-AP, 1:10000; Proteintech), and histone H3 (4499, 1:1000; Cell Signaling Technology).
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