Cellular proteins were extracted using radio immunoprecipitation assay (RIPA) lysis buffer, and protease inhibitors were added to the lysate to protect the proteins. Anti-FTH polyclonal antibody (ab65080, Abcam), anti-αS1002/S100A2 (ab109494, Abcam), S100A4 (ab124805, Abcam), and S100P (ab133554, Abcam) monoclonal antibodies were diluted as the first antibody in a 1:1,000 ratio. Anti-mouse IgG H&L horseradish peroxidase (HRP), goat anti-rabbit IgG H&L HRP, and mouse anti-β actin monoclonal antibodies (Beijing Dingguo Chang Sheng Company) were diluted to a 1:2,500 ratio. Primary antibodies and polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4 °C. The next day, the PVDF membranes were washed 3 times in phosphate-buffered saline (PBS) buffer. Then, they were incubated with secondary antibodies, which were labeled with HRP for 1 h. Target protein bands were determined by enhanced chemiluminescence, and β-actin was an internal reference to achieve consistent sample loading per well. Each experiment was repeated 3 times.
Ab124805
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab124805 is an immunohistochemistry antibody produced by Abcam. It is designed for the detection of target protein in specific tissue or cell types.
Automatically generated - may contain errors
Lab products found in correlation
Ab92547, by Abcam (4 mentions)
Ab32575, by Abcam (4 mentions)
Ab8245, by Abcam (3 mentions)
Ab53066, by Abcam (3 mentions)
Ab195380, by Abcam (2 mentions)
Ab125011, by Abcam (2 mentions)
Ab207178, by Abcam (2 mentions)
Ab124964, by Abcam (2 mentions)
Ab193555, by Abcam (2 mentions)
Goat anti rabbit envision system plus hrp, by Agilent Technologies (2 mentions)
Ab133626, by Abcam (2 mentions)
Ab109494, by Abcam (1 mentions)
Ab65080, by Abcam (1 mentions)
Ab133554, by Abcam (1 mentions)
Ab15007, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab124892, by Abcam (1 mentions)
Ab195377, by Abcam (1 mentions)
Ab288151, by Abcam (1 mentions)
19 protocols using ab124805
1
Protein Extraction and Immunoblotting
2
Immunohistochemical Characterization of Skin Tissues
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Tissues of normal skin and keloid were fixed in formalin for 48 h, embedded, and sectioned at a thickness of 4 μm. Then, the sections were deparaffinized, rehydrated, and blocked with phosphate-buffered saline (PBS) or 2% fetal bovine serum (FBS). Sections were incubated overnight at 4°C with specific primary antibody rabbit anti-S100A4 (1:500, Abcam, ab124805) and rabbit anti-WTAP (1:500, Abcam, ab195380) and then incubated with the secondary antibody goat anti-rabbit IgG (1:1,000, Abcam, ab15007) at 37°C for 30 min in dark. Subsequently, the sections were washed with PBS three times (5 min each time). Finally, the sections were counterstained with DAPI (1 μg/ml, Abcam, ab285390-5 mg) to allow visualization of the cell nucleus.
3
Western Blot Analysis of RNA Methylation Regulators
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Tissues were homogenized in RIPA lysis buffer containing a 50× protease inhibitor cocktail. Homogenates were then centrifuged at 19,392 g for 15 min to remove cell debris. The supernatant was collected, and total protein concentrations were measured using a protein assay kit (Enhance6d BCA Protein Assay Kit, Beyotime). A volume of 10 μg of the protein was electrophoretically separated in an SDS-PAGE gel (10% Tris–HCl) and transferred to 4.5-μm PVDF membranes. The membranes were blocked with 5% skim milk for 1 h and then incubated with rabbit anti-Mettl3 (1:1,000, ab195352, Abcam), rabbit anti-Mettl14 (1:1,000, ab220031, Abcam), rabbit anti-WTAP (1:1,000, ab195380, Abcam), rabbit anti-β-catenin (1:1,000, #8480, Cell Signaling Technology), rabbit anti-Wnt3 (1:1,000, #2721, Cell Signaling Technology), rabbit anti-S100A4 (1:1,000, ab124805, Abcam), rabbit anti-FTO (1:1,000, ab124892, Abcam), rabbit anti-ALKBH5 (1:1,000, ab195377, Abcam), and mouse anti-GAPDH (1:1,000, ab8245, Abcam) overnight at 4°C room temperature. The membranes were then washed in PBS-T (3 times for 10 min each) and incubated with the anti-rabbit secondary antibody (1:1,000, ab288151, Abcam) or anti-mouse secondary antibody (1:1,000, ab150113, Abcam) at room temperature for 1 h. Bands were visualized by ECL and quantitated using ImageJ software.
4
Western Blot Analysis of Protein Markers
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Briefly, cells were lysed in RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Nantong, China) at 48 hr following transfection as described below and protein concentration was determined by the BSA method (Beyotime Institute of Biotechnology). Equivalent quantities of protein were separated on a 10% SDS‐polyacrylamide gels and then transferred to Polyvinylidene Fluoride (PVDF) Membrane. Membranes were blocked using 5% non‐fat milk and incubated overnight with the appropriate primary antibody. The next day, they were washed three times with TBST and incubated with a HRP‐conjugated secondary antibody (Beyotime Institute of Biotechnology) at 1:5,000 dilution for 1 hr at room temperature. Protein detection was performed using the enhanced chemiluminescence (ECL) system (Millipore, Bedford, MA). Primary immunoblotting antibodies were: anti‐β‐Actin(dilution 1:1,000, 4,970, Cell Signaling Technology, Danvers, MA), anti‐DPP9(dilution 1:1,000, ab42080, Abcam, Cambridge, MA), anti‐p53 (Epitomics, Burlingame, CA), anti‐BAX(dilution 1:1,000, ab32503, Abcam), anti‐APAF1(dilution 1:1,000, ab32372, Abcam), anti‐MUC1(dilution 1:1,000, ab45167, Abcam), anti‐S100A4(dilution 1:1,000, ab124805. Abcam), anti‐E‐caderin(1:50, ab1416), anti‐vimentin (1:1,000, ab92547, Abcam).
5
Comprehensive Protein Expression Analysis
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Antibodies against CD63 (ab125011, 1:1000), α-SMA (ab32575, 1:100), FAP (ab207178, 1:1000), FSP (ab124805, 1:1000), GAPDH (ab8245, 1:10,000), CD9 (ab92726, 1:1000), CD81 (ab79559, 1:1000), FASN (ab22759, 1:500), ATP citrate lyase (EP704Y, 1:1000) and phospho-ATP citrate lyase (T447/S451) (ab53007, 1:1000) and USP2 (ab66556, 1:500) were purchased from Abcam (Cambridge, MA, USA). Antibodies against phosphor-PTEN (Ser380/Thr382/383) (9554S, 1:1000), total PTEN (7960 T, 1:1000), PDK1 (3062 T, 1:1000), phospho-PDK1 (Ser241) (3438 T, 1:1000), phospho-Akt (Ser473) (4060 T, 1:1000) and AKT (4691 T, 1:1000) were supplied by Cell Signaling Technology (Beverly, MA, USA). AKT inhibitor MK-2206 was provided by Selleck (Houston,USA).
6
Western Blot Analysis of Fibrosis Markers
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We performed western blot (WB) analysis as previously described36 using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).
7
Immunofluorescence Staining of Cultured Cells
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Cells were cultured on four-well chamber slides. At the time of harvest, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.01% Triton X-100 for 10 min. Then cells were treated with anti-α-SMA antibody (Abcam, ab124964), anti-FAP antibody (Abcam, ab53066), and anti-FSP1 (Abcam, ab124805). In addition, all samples were treated with 40, 6-diamidino-2-phenylindole dye for nuclear staining (358 nm). For confocal microscopy, a Nikon C2 Plus confocal microscope was used.
8
Protein Quantification and Western Blot Analysis
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Tissues and cells were lysed in RIPA buffer (cat. no. R0020; Solarbio Science and Technology, Beijing, China) to recover total protein. Then the protein quantification was measured using a bicinchoninic acid assay kit (cat. no. 23227; Thermo Fisher Scientific). Twenty micrograms of protein was loaded onto SDS‐PAGE and transferred to polyvinylidene difluoride membranes (cat. no. 1620177; Bio‐Rad). After blocking, protein levels were detected by specific primary antibodies from Abcam (Cambridge, MA, USA) against PDPN (ab236529, 1:1000), ezrin (ab4069, 1:500), Ki67 (ab16667, 1:1000), E‐cadherin (ab40772, 1:10000), N‐cadherin (ab76011, 1:1000), cleaved‐caspase 3 (ab2302, 1:5000), α‐smooth muscle actin (α‐SMA; ab5831, 1:1000), fibroblast activation protein (FAP; ab207178, 1:1000), fibroblast‐specific protein 1 (FSP‐1; ab124805, 1:1000) and GAPDH (ab8245, 1:2500), followed by the corresponding horseradish peroxidase‐conjugated secondary antibodies (ab6721/ab205719, 1:5000) and visualization. GAPDH was used as the internal control.
9
Comprehensive Western Blotting Protocol
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Western blotting was performed as previously described 18 (link). Briefly, equal amounts of total protein per sample were separated on a polyacrylamide gel containing 0.1% SDS and then transferred to PVDF membrane. The membranes were incubated overnight with primary antibodies of TRPC5 (1:1000, ACC-020, Alomone Labs), β-actin (1:1000, #4967, Cell Signaling Technology), TSG101 (1:1000, ab125011, Abcam), CD81 (1:1000, ab109201, Abcam), fibroblast activation protein (FAP) (1:1000, ab53066, Abcam), a-SMA (1:1000, ab32575, Abcam), FSP-1 (1:1000, ab124805, Abcam) and Vimentin (1:1000, ab193555, Abcam) at 4°C, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:500, ab205718, Abcam) for 1 h at room temperature. The protein bands were developed with an enhanced chemiluminescence kit (E411-04, Vazyme), and observed using ChemiDocXRS+ gel imager (Bio-Rad).
10
Immunofluorescence Staining for Cell Markers
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Cells were spread into 12-well plates on glass cover-slides. When cell confluence reached to 70%, the supernatant was discarded and 4% paraformaldehyde was fixed for half an hour. Primary antibodies of α-SMA (Abcam, ab32575), S100A4 (Abcam, ab124805), and vimentin (Abcam, ab92547) were incubated at 4 °C overnight. Cells were washed for 3 times with PBS on the next day and incubated with corresponding second antibody at room temperature for 1 h. Cell slides were collected and sealed by DAPI solution (Abcam, ab104139).The images were observed under a confocal microscope (Leica, Germany).
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