The proteins from hASCs from the experimental and control groups at day 7, day 14, and day 21 were extracted using the cell protein extraction kit. The cells in each dish were lysed in 100 mL lysis buffer (CWBIO, Beijing, China) containing 1 mM phosphorylated protease inhibitor (Thermo, USA), and the samples were incubated on ice for 20 minutes. The extracted proteins were detected by BCA analysis and were loaded on 10% or 12% SDS-PAGE gel electrophoresis. The samples were transferred to a PVDF membrane and incubated with blocking solution of TBST containing 0.05 g/mL bovine serum albumin (BSA) for one hour. After blocking, the samples were incubated with their corresponding primary antibodies overnight at 4°C. The primary antibodies included OPN (1: 2,000, Abcam), OCN (1: 1,000, Abcam), and RUNX-2 (1: 1,000, Abcam). After washing the membrane three times in TBST, the membrane was incubated with the corresponding secondary antibody for one hour. After washing the membrane three times in TBST, the immunoreactive band was detected with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The optical densities of the bands were quantified using ImageJ (Scion Corporation).
Imagej
Manufactured by Techcomp Instruments
Sourced in United States
ImageJ is an open-source image processing software designed for scientific use. It provides a wide range of tools for image analysis, processing, and measurement. ImageJ's core function is to enable users to view, edit, analyze, process, save, and print 8-bit, 16-bit, and 32-bit images.
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Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (8 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Phosstop, by Roche (2 mentions)
Ecl plus system, by GE Healthcare (2 mentions)
Mayer s hematoxylin, by Carl Roth (2 mentions)
Nf κb p65, by Abcam (2 mentions)
α sma, by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by CWBIO (1 mentions)
Runx2, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
It 500hr, by JEOL (1 mentions)
Bca protein kit, by Beyotime (1 mentions)
Bca assay kit, by Nanjing Jiancheng (1 mentions)
Tgf β1, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Collagen 1, by Cell Signaling Technology (1 mentions)
29 protocols using imagej
1
Protein Expression Analysis of hASCs
2
Western Blot Analysis of Autophagy Markers
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After each treatment, the cells were lysed in RIPA buffer with protease inhibitor cocktail (Roche Applied Bioscience, Indianapolis, IN, USA) and subjected to western blotting as described previously (Rao et al., 2016 (link)). Briefly, protein concentrations were assessed with a BCA Kit (Pierce, Rockford, IL, USA), and equivalent amounts of protein (30 μg) were separated by 8–15% SDS-PAGE gels, followed by transfer onto PVDF membranes. The membranes were then soaked in 5% skim milk in Tris-phosphate buffer containing 0.05% Tween 20 (TBST) for 1 h and further incubated overnight at 4°C with the appropriate primary antibody (Homer1a 1:1000; LC3II 1:1000; Beclin-1 1:1000; P62 1:8000; β-actin 1:5000; AMPK 1:1000; p-AMPK 1:1000). After three washes for 8 min in TBST, the blots were incubated with HRP-conjugated secondary antibodies for 1–2 h. The target protein signal was detected by SuperSignal West Pico Chemiluminescent Substrate (Thermos Scientific). The optical densities of the bands were quantified by ImageJ (Scion Corporation, Torrance, CA, USA).
3
Morphological Analysis of Hyaluronic Acid Hydrogels
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The morphology of native HA, cross-linked HA and MXF-HA was visualized by SEM using an IT-500HR instrument (JEOL, Tokyo, Japan) as previously described16 (link). All samples were lyophilized prior to processing. Images were taken in a Secondary Electron Imaging (SEI) mode with a voltage of 15 kV. Each sample solution was frozen overnight, then immersed in liquid nitrogen for 1 h, and the samples were fixed to aluminum plates with carbon tape and then coated by platinum sputtering. The pore size of the honeycomb structure of MXF-HA was measured using image analysis software (ImageJ, Scion Corp., Frederick, Maryland, USA).
4
Regenerating Muscle Fiber Analysis
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Photomicrographs of regenerating muscle fibers (identified by morphological criteria, i.e., centrally located nuclei in H&E stained sections) were taken at standard resolution (1.300 × 1030 pixel) and analyzed using ImageJ, Scion Image software. For morphometric evaluation of fiber size, 200–1000 cross-sectioned fibers per sample were analyzed. Quantitative data were obtained from three independent experiments in triplicate. The values are expressed as mean ± SD.
5
Protein Expression Analysis in Metabolic Regulation
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Equivalent amounts of protein (40 μg per lane) were loaded and separated by 10% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skimmed milk solution in tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h, and then incubated overnight at 4 °C with the primary Sirt1 (1:1000), Sirt3 (1:1000), p-AMPK (1:400), AMPK (1:600), PGC1 (1:600), cytochrome c (1:800), 3-NT (1:800), COX V (1:600), Tubulin (1:1000) or β-actin (1:1000) antibody dilutions in TBST. After that the membranes were washed and incubated with secondary antibody for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Image J (Scion Corporation) was used to quantify the optical density of each band.
6
Quantitative Western Blot Analysis of Endocannabinoid Signaling
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After various treatments, HT22 cells in 6-cm dishes were washed three times with ice-cold PBS and lysed with lysis buffer containing protease inhibitor mixture tablets and phosphatase inhibitor mixture tablets (PhosSTOP, Roche Applied Science, Mannheim, Germany). Protein concentration of the supernatant was determined using a BCA protein kit (Beyotime, Shanghai, China). Proteins were separated by 10–15% and 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Thermo Scientific). The membranes were soaked in 5% nonfat milk solution in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with the appropriate primary antibody (CB1, 1:800; DAGL, 1:800; NAPE-PLD, 1:500; MAGL, 1:500; FAAH, 1:800; p-ERK, 1:800; ERK, 1:1000; β-actin, 1:2500). Membranes were washed in TBST and incubated for 1 h at room temperature with the secondary antibodies diluted in blocking buffer. Immunoreactivity was detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Optical densities of the bands were quantified using an image analysis system with ImageJ (Scion Corporation).
7
Western Blot Analysis of Spinal Cord Proteins
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Total proteins from spinal cord tissues were extracted and the protein concentration was determined using a BCA assay kit (Jiancheng Biotech Company, Jiangsu, China). Equivalent proteins (60 μg per sample) were separated using 10 or 12% sodium dodecyl sulfate (SDS)-PAGE, and then electro-transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with the following primary antibodies: SK/KCa1 (1:500), COX IV (1:800), Tubulin (1:1000), Opa-1 (1:500), Mfn-1 (1:800), Drp-1 (1:800), Fis-1 (1:500), and β-actin (1:2000). After incubation with secondary antibodies for 1 h, the bands were visualized by using chemiluminescent detection system. Image J (Scion Corporation) was used to quantify the optical density of each band. The expression of each protein was calculated from the optical density of each band normalized against the optical density of β-actin, tubulin or COX IV, and expressed as the fold of control levels.
8
Western Blot Analysis of Protein Expression
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Equivalent amounts of protein (30 μg per lane) were loaded and separated by 12% SDS-PAGE gels and transferred electrophoretically to PVDF membranes. Following protein transfer to PVDF membranes, the membranes were blocked and then incubated overnight at 4 °C with specific primary antibodies as follows: SIRT1, HIF-1α, SIRT3, p62, LC3, SOD2, TGF-β1, collagen-1, Smad-3 and β-actin were obtained from Cell Signaling Technology, Inc. (Beverly, Mass.). The bands of proteins were visualized using the ECL Plus system (GE Healthcare, Little Chalfont, United Kingdom). Image J (Scion Corporation) was used to quantify the optical density of each band.
9
Quantifying Myocardial Infarction Size
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The infarct size was determined by calculating the percentage of the infarcted area against the whole LV area using Scion ImageJ. The peri-infarct regions from the MI model rats and cell therapy rats were embedded in paraffin, sectioned, and stained with triphenyltetrazolium chloride (TTC), hematoxylin and eosin (H&E), and Masson's trichrome or by immunohistochemistry or immunofluorescence.
10
Western Blot Analysis of EMT Markers
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Equivalent amounts of protein (30 μg per lane) were loaded and separated by 8%-12% SDS-PAGE gels, and transferred PVDF membrane. After the transfer was completed, the membrane was blocked 30 min and then incubated overnight at 4° with specific primary antibodies as follows: antibodies against E-Cadherin, Cytokeratin, Vinmentin, N-Cadherin and CRKL were obtained from Abcam, β-actin was purchased from Biyuntian Biotechnology Co., LTD. After that the membranes were washed and incubated with secondary antibody for 1 h at room temperature. The bands of proteins were visualized using the ECL Plus system (GE Healthcare, Little Chalfont, United Kingdom). Image J (Scion Corporation) was used to quantify the optical density of each band.
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