The optical density of the western blot signals was quantified using the National Institutes of Health ImageJ software and all statistical analyses were carried out using the SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL, USA). The expression level of p53 was quantified relative to β-actin and the differences between the cancer tissues and adjacent normal tissues in p53 protein expression levels were compared using Student’s t-test. The χ2 test was performed to evaluate the correlation between the clinicopathological features of the patients and the p53 expression level, which was observed by IHC. Kaplan-Meier survival analysis was used to evaluate the patient prognosis and the eight-year survival rate of the ESCC patients was obtained using the life table method. A univariate analysis was plotted using the Kaplan-Meier method and Cox regression analysis was used to evaluate the correlation between risk of ESCC and clinicopathological parameters, including p53 expression. P≤0.05 was considered to indicate a statistically significant difference.
Imagej software
Manufactured by IBM
Sourced in United States
ImageJ is an open-source software for image processing and analysis. It provides a wide range of tools for image manipulation, measurement, and analysis. ImageJ is primarily used for scientific research and medical imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Spss 13.0 statistical software package, by IBM (1 mentions)
Spss statistics v22, by IBM (1 mentions)
Ab191606, by Abcam (1 mentions)
Ab38449, by Abcam (1 mentions)
Gel imager system, by Bio-Rad (1 mentions)
Ab179450, by Abcam (1 mentions)
Ab109543, by Abcam (1 mentions)
Ab40777, by Abcam (1 mentions)
Ab259337, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab178860, by Abcam (1 mentions)
Ab40761, by Abcam (1 mentions)
Sc 2048, by Bio-Rad (1 mentions)
Ab9485, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Af7021, by Affinity Biologicals (1 mentions)
Ecl chemiluminescent kit, by Biosharp (1 mentions)
5 protocols using imagej software
1
Evaluation of p53 Expression in ESCC
2
Image Analysis and Statistical Methods
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Results were analyzed using Image J software and SPSS Statistics V 22.0 (SPSS, Inc., Chicago, IL, United States). Multiple group comparisons were performed using Student’s t-test or ANOVA with Tukey’s posttest. P < 0.05 was considered to indicate a statistically significant difference.
3
Evaluating Esophageal Tissue Ablation
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Esophageal tissue specimens obtained from euthanized swine were removed en bloc, followed by gross inspection. Each specimen was photographed, and the ablation outcomes—degree of erythema, erosion, and ulceration size—were configured using ImageJ software (IBM). Next, the specimens were fixed in 10% formalin for 1 day, excised at a thickness of 3 µm, and examined by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay to analyze the depth of ablation. The tissue depth at which the most damaged cells were found was identified. The ablation area and depth were calculated as the average of the two lesions produced during esophageal ablation.
4
Western Blot Analysis of Insulin Signaling Proteins
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Radioimmunoprecipitation assay buffer (RIPA) (Sangon, China) was used to extract
proteins from cultured cells and the concentration was determined using a
bicinchoninic acid kit (Sangon Biotech Co., Ltd.). Total protein (10 µg) was
resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF)
membranes. Non-fat milk (10%) in PBS buffer (90%) was used to block the
membranes at room temperature for 1 h. Then, the membranes were incubated with
primary antibody at 4°C overnight and secondary antibody at room temperature for
2 h. The immunoreactive bands were developed using an enhanced chemiluminescence
kit (Santa Cruz, USA, sc-2048) and photographed on a gel imager system (Bio-Rad,
USA). The densitometry analysis was performed with ImageJ software (IBM, USA).
The antibodies used in this study were as follows (all from Abcam, USA): GAPDH
(1:2500; ab9485; USA); C/EBP-α (1:1000; ab40761); PPAR-γ (1:1000; ab178860);
C/EBP-α (1:1000; ab40761;); IRS (1:10000; ab40777); p-IRS (1:10000; ab109543);
AKT (1:500; ab8805); p-AKT (1:500; ab38449); FoxO1 (1:1000; ab179450); p-FoxO1
(1:1000; ab259337); PI3K (1:1000; ab191606); p-PI3K (1:1000; ab278545); β-actin
(1:1000; ab8226). In addition, HRP-linked secondary antibody was used (1:3000;
Cell Signaling #7074, USA).
proteins from cultured cells and the concentration was determined using a
bicinchoninic acid kit (Sangon Biotech Co., Ltd.). Total protein (10 µg) was
resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF)
membranes. Non-fat milk (10%) in PBS buffer (90%) was used to block the
membranes at room temperature for 1 h. Then, the membranes were incubated with
primary antibody at 4°C overnight and secondary antibody at room temperature for
2 h. The immunoreactive bands were developed using an enhanced chemiluminescence
kit (Santa Cruz, USA, sc-2048) and photographed on a gel imager system (Bio-Rad,
USA). The densitometry analysis was performed with ImageJ software (IBM, USA).
The antibodies used in this study were as follows (all from Abcam, USA): GAPDH
(1:2500; ab9485; USA); C/EBP-α (1:1000; ab40761); PPAR-γ (1:1000; ab178860);
C/EBP-α (1:1000; ab40761;); IRS (1:10000; ab40777); p-IRS (1:10000; ab109543);
AKT (1:500; ab8805); p-AKT (1:500; ab38449); FoxO1 (1:1000; ab179450); p-FoxO1
(1:1000; ab259337); PI3K (1:1000; ab191606); p-PI3K (1:1000; ab278545); β-actin
(1:1000; ab8226). In addition, HRP-linked secondary antibody was used (1:3000;
Cell Signaling #7074, USA).
5
Western Blot Analysis of RBMS3 Protein
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A lysis buffer containing 1 mM phenylmethylsulfonate fluoride (PMSF; No.: BL612A; Beijing Labgic Technology Co., Ltd., China) was applied to extract the total protein using RIPA (No.: P0013B; Shanghai Beyotime Biotech Co., Ltd., China). Using a fast preparation kit (No.: ZJ102; Shanghai Yazy Biomedical Technology Co., Ltd., China), after samples were electrophoretically separated on 10% SDS-PAGE gels, they were transferred to PVDF membranes for 80 min at a constant current of 200 mA. Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes. After that, they had three TBST washes (10 min each wash). The following step involved incubating an anti-rabbit secondary antibody coupled with horseradish peroxidase for 1 h under a condition of the room temperature. After that, the PVDF membranes were found using the ECL-chemiluminescent kit (No.: BL520A, Biosharp, Beijing, China) and quantified using ImageJ software from IBM Corp. (Armonk, NY, USA).
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