NRK-52E cells that had undergone various treatments were fixed in 4% paraformaldehyde and per-mobilized in 0.1% Triton X-100, before they were, respectively, treated with primary mouse monoclonal anti-E-cadherin (E-ca) antibody (Abcam ab1416, 1:100), mouse monoclonal collagen I antibody (Abcam ab260043, 1:100), TLR4 (Abcam ab22048, 1:200), NF-κB (Abcam ab194726, 1:200). After three washes with PBS, the sections were incubated for 2 h with DAR-FITC (1:50) and Texas Red-DAM (1:50) at RT. The fluorescent images were visualized with a Fluoview 300 fluorescence microscope (Olympus, Tokyo, Japan).
Ab260043
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab260043 is a laboratory reagent. It is designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab124964, by Abcam (10 mentions)
Ab181602, by Abcam (8 mentions)
Ab7778, by Abcam (7 mentions)
Ab7817, by Abcam (6 mentions)
Ab8245, by Abcam (6 mentions)
Ab2413, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ab150077, by Abcam (5 mentions)
Ab215715, by Abcam (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ab5694, by Abcam (4 mentions)
Ab8226, by Abcam (4 mentions)
Ab268020, by Abcam (3 mentions)
Ab45688, by Abcam (3 mentions)
Ab39012, by Abcam (3 mentions)
Ab181286, by Abcam (3 mentions)
Ab182858, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Ab236639, by Abcam (3 mentions)
Ab179483, by Abcam (3 mentions)
72 protocols using ab260043
1
Immunofluorescence Imaging of Cellular Markers
2
Protein and Mitochondrial Profiling by Western Blot
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The relative expression of total protein or mitochondrial protein were detected by western blot. Antibodies used here were as follows: Collagen I (ab260043, abcam), α-SMA (ab7817, abcam), VDAC1 (A5224, Bimake), p62 (18420-1-AP, proteintech), LC3 II/I (A5179, bimake), SIRT1 (13161-1-AP, Proteintech), GAPDH (RM2001; Ray Antibody Biotech), and secondary antibodies (92632210, 92632211, Licor). The bands were visualized by Odyssey System (LI-COR).
3
Comprehensive Protein Expression Analysis
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Total protein was collected according to the methods described in our previous report. The protein concentration was assessed using a Thermo Fisher bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies diluted to the recommended concentration, including mouse anti-α-SMA (Abcam, Ab240654, 1:500), rabbit anti-collagen I (Abcam, Ab260043 1:500), rabbit anti-SENP1 (Abcam, Ab236094, 1:500), rabbit anti-β-Catenin (Abcam, Ab32572, 1:500), and rabbit anti-GLI1 (Abcam, Ab217326, 1:500), rabbit anti-SPC (Abcam, Ab211326, 1:500) overnight at 4 °C. The membranes samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 h, and the protein bands were detected using a chemiluminescence device.
4
Comprehensive Protein Expression Analysis
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Total protein from cells was extracted with RIPA buffer (abs9229, Absin, China). The extracted protein was centrifuged and then quantified with the BCA kit (pc0020, Solarbio, China). After electrophoresis, they were electrotransferred onto the nitrocellulose membrane. Then, 5% nonfat milk was taken to block the membrane. After rinsing, the blocked membrane was subjected to primary antibodies at 4°C all night. The next day, an antirabbit secondary antibody (ab7090, Abcam, UK) was added. After reacting at 37°C for 1 h, a color reagent (1705061, BIO-RAD, USA) was taken to visualize the blots. Finally, the blots were developed in ChemiScope 3300 mini equipment (Clinx, China). The primary antibodies of collagen II (1: 1000, ab34712), fibronectin (1: 1000, ab268020), α-SMA (1: 50000, ab124964), Notch1 (1: 2000, ab52627), Jag1 (1: 500, ab7771), HEY1 (1: 3000, ab154077), HES1 (1: 1000, ab108937), TGF-β (1: 1000, ab215715), E-cadherin (1: 50000, ab40772), vimentin (1: 5000, ab92547), N-cadherin (1: 20000, ab76011), collagen I (1: 1000, ab260043), and GAPDH (ab181602) were bought from Abcam (UK).
5
Western Blot Analysis of Fibrosis Markers
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The expression levels of α-SMA, COL1α1, FN1, and vimentin were examined by WB. The following antibodies were used: anti-α-SMA (Abcam, ab124964), anti-COL1α1 (Abcam, ab260043), anti-FN1 (Abcam, ab45688), anti-vimentin (Abcam, ab92547) and anti-β-actin (Immunoway, YM3028). Details are provided in the Additional file 2 : Methods.
6
Western Blot Analysis of Osteogenic Markers
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Total cellular protein was isolated from the cells using the radio-immunoprecipitation assay (RIPA) buffer (Beyotime) containing protease inhibitors at 4 ºC for 30 min. Protein concentrations were measured with BCA protein, separated by SDS-PAGE (30 µg/well), and transferred to polyvinylidene fluoride membranes and blocked. The membranes were incubated with primary antibodies including ALP (ab229126, Abcam), OPN (ab283656, Abcam), RUNX2 (ab23981, Abcam), Colla1 (ab260043, Abcam), and KDM1A (ab62582, Abcam). Afterward, these membranes were washed and followed by incubation with a secondary antibody (ab7090, Abcam). Eventually, an ECL kit (Millipore) was utilized to visualize the protein bands.
7
Immunohistochemical Staining of Lung Sections
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Immunohistochemical staining was performed on paraffin embedded lung sections. The sections were deparaffinized with xylene and prepared for staining as described previously. Sections were blocked with 10% goat serum in PBS for 1 h, followed by incubation with the following primary antibodies at 4 °C for 12 h: rabbit anti-mouse α-SMA (Abcam, ab124964, dilution 1:500) and rabbit anti-mouse COL1A (Abcam, ab260043, dilution 1:500). After washing, sections were incubated with biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (DBA) at room temperature for 2 h, and photographed using a light microscope (Motic, China) at the magnification of × 100. To assess nonspecific staining for α-SMA and COLA1, alternate sections from each experimental condition were also incubated with primary or secondary antibody only [20 (link)].
8
Western Blot Analysis of Protein Markers
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Western blot was performed as described in our previous studies [2 (link)]. In brief, total protein was extracted using pre-cooled RIPA lysis buffer (Beyotime, Shanghai, China) containing PMSF and phosphatase inhibitor, and then total protein concentration was measured by a BCA protein quantification kit (Beyotime, Shanghai, China). After denaturation, equal amounts of total protein were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Subsequently, the PVDF membrane was sealed with 5% skim milk for 1 h, and then incubated with primary antibodies for overnight at 4 °C. After overnight incubation of the primary antibody, the PDVF membrane was incubated with the secondary antibody for 2 h at room temperature. Finally, immunoreactive protein bands were visualized and analyzed by the ECL kit (Pierce, Thermo Fisher Scientific, IL, USA) and ImageJ. Primary antibody information was as follows: cleaved caspase-3 (1:500, ab32042, Abcam), Bax (1:1000, ab182733, Abcam), Bcl-2 (1:500, ab182858, Abcam), VEGFA (1:1000, ab214424), collagen I (1:1000, ab260043, Abcam), collagen III (1:1000, ab7778, Abcam), MMP2 (1:500, ab181286, Abcam), MMP13 (1:1000, ab39012, Abcam), GAPDH (1:1000, ab8245, Abcam), and β-actin (1:2000, ab8227, Abcam).
9
Immunofluorescence Staining of A549 Cells
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A549 cells in the logarithmic growth phase were collected and ~1×105 cells/per well were placed in immunofluorescence dishes until they had adhered to the plate. The old culture medium was discarded. Following TGF-β1 stimulation and/or cell transfection for 48 h, the A549 cells were washed with pre-cooled PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the A549 cells were permeated for 10 min with 0.5% Triton X-100 at room temperature. Normal goat serum was added into the dish and incubated at room temperature for 30 min. After the blocking reagent was discarded, the appropriate primary antibodies collagen I (ab260043; 1:250; Abcam), α-SMA (ab124964; 1:250; Abcam) and E-cadherin (ab40772; 1:500; Abcam) were added to each dish and placed in a wet box for incubation at 4°C overnight. The cells were then incubated at room temperature for 1 h in a wet box with a fluorescent secondary antibody (goat anti-rabbit IgG H&L; Alexa Fluor® 488; dilution, 1:500; ab150077; Abcam). DAPI was added to the A549 cells and incubated for 5 min in the dark to stain the cell nuclei. Anti-fluorescence quenching agent was added to the dishes, and images were then captured using an inverted fluorescence microscope (Olympus Corporation) at a magnification of ×200.
10
Protein Expression Analysis in Bone Cells
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Briefly, total proteins from MG63 and U2OS cells were isolated using radio-immunoprecipitation assay (RIPA) lysis buffer and protein concentration was quantified using a bicinchoninic acid (BCA) Protein Assay kit. Equal amount of protein samples (30 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. Nonspecific binding proteins were blocked with 5% nonfat milk, primary antibodies against MMP2 (Abcam, ab181286, 1:1000), MMP9 (Abcam, ab228402, 1:1000), Collagen 1 (Abcam, ab260043, 1:1000), osteopontin (Abcam, ab214050, 1:1000), RANKL (Abcam, ab65024, 1:1000), Runx2 (Abcam, ab23981, 1:1000), osteocalcin (Abcam, ab181286, 1:5000), DKK1 (Abcam, ab93017, 1:1000), β-catenin (Abcam, ab224803, 1:1000), PTHR1 (Abcam, ab189924, 1:1000), and GAPDH (Abcam, ab181602, 1:1000) were applied to incubate membranes overnight at 4°C. On the next day, secondary antibodies (Abcam, ab205718, 1:50,000) were employed to incubate membranes for 1.5 h at room temperature. Protein bands were developed with electrochemiluminescence (ECL) detection kit (Beyotime, Shanghai, China). Protein expression was analyzed using Image J software with GAPDH as the internal reference.
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