The cells were washed twice with PBS, permeabilized in 0.1% Triton X-100, neutralized with NH4Cl buffer and then per-meabilized using 0.05% saponin/PBS (pH 7.4) for 45 minutes. Subsequently, the samples were incubated overnight with the following primary antibodies: cyt-c (1:500; Abcam, #ab90529), Drp1 (1:1,000; Abcam, #ab56788) and Tom-20 (1:1,000; Abcam, #ab186735). Confocal immunofluorescence images were collected using the FV10-ASW 1.7 software and an Olympus IX81 microscope.24 The fluorescence intensity was calculated using the Image-Pro Plus 6.0 software. First, fluorescence pictures were converted to grayscale with the Image-Pro Plus 6.0 software. Then, the fluorescence intensities were separately recorded as the grayscale intensities. Mitochondria were observed in at least 100 cells, and the average length of the mitochondria was measured under an inverted microscope to quantify mitochondrial fragmentation (BX51; Olympus Corporation, Tokyo, Japan) as described in a previous study.25 (link) The experiments were performed in triplicate and repeated three times with similar results.26 (link)
Ab90529
Manufactured by Abcam
Sourced in United States
Ab90529 is a lab equipment product offered by Abcam. It is a device used for specific purposes in a laboratory setting. The core function of this product is to facilitate certain experimental or analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab186735, by Abcam (7 mentions)
Cyt c, by Abcam (4 mentions)
Ab24170, by Abcam (3 mentions)
Ab2302, by Abcam (3 mentions)
Ab53154, by Abcam (2 mentions)
Ab2324, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Sc 47778, by Santa Cruz Biotechnology (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
H2o2 solution, by Merck Group (1 mentions)
Streptomycin, by Corning (1 mentions)
Penicillin, by Corning (1 mentions)
Ab16056, by Abcam (1 mentions)
Ab181591, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab80588, by Abcam (1 mentions)
Ab33911, by Abcam (1 mentions)
Ab185002, by Abcam (1 mentions)
Ab82419, by Abcam (1 mentions)
Ab118285, by Abcam (1 mentions)
17 protocols using ab90529
1
Immunofluorescence Analysis of Mitochondrial Dynamics
2
Phillyrin Modulates Oxidative Stress and Apoptosis in ARPE-19 Cells
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Human ARPE-19 cell line was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Phillyrin (batch number Z17A8X34077, purity > 98%) was a product of the China Food and Drug Administration (Beijing, China). Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, and streptomycin solutions were purchased from Corning (NY, USA). Trypsin (0.05%) and phosphate buffered saline (PBS) were produced by Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT), ML385, N-acetylcysteine (NAC)m and H2O2 solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Fas (ab82419), cytochrome c (ab90529), cleaved caspase-3 (ab2302), cleaved caspase-9 (ab2324), NQO1 (ab80588), Keap1 (ab118285), Bcl-2 (ab185002), Nrf2 (ab62352), CDK2 (ab32147), cyclin A (ab33911), cyclin E (ab181591), Bax (ab53154), β-actin (ab8226), p21 (ab188224), Histone H3 (ab1791), COX IV (ab16056) p53 (ab241556), pro caspase-3, pro caspase-8, and pro caspase-9 were obtained from Abcam, Cambridge, (MA, USA). Antibodies against p-p53 (9286S), cleaved caspase-8 (9496S), cleaved PARP (5625S), and HO-1 (86806S) were purchased from Cell Signaling Technology, Beverly, (MA, USA).
3
Immunofluorescence Staining of Organelles
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Cells were plated on glass slides in a 6-well plate at a density of 1 × 106 cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature. The cells were then incubated with the primary antibodies: [cyt-c (1:1000; Abcam; #ab90529), Tom20 (mitochondrial marker, 1:1000, Abcam, #ab186735), LAMP1 (lysosome marker, 1:1000, Abcam, #ab24170), MIEF1 (1:1000, Abcam, #ab89944)] [39 (link)].
4
Apoptosis Regulation in NP-MSCs
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The cells were lysed by using a western and IP cell lysis kit. After NP-MSCs were treated with 100 μM pioglitazone or 1.0 MPa compression, apoptosis-related proteins were detected by western blotting. Also, to determine the cytochrome c of cytosol (cyto.cytochrome c), the cytosol protein of cells was isolated according to the manufacturer's instructions of the Cell Mitochondria Isolation Kit (Beyotime, China). The protein concentration was measured by using the BCA protein assay kit (Beyotime, China). Equal protein amounts (30 μg) were resolved on 10%-12% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore, Burlington, MA, USA), then the membranes were blocked by 5% nonfat milk and then incubated overnight at 4°C with primary antibodies against cytochrome c (1 : 1000, Abcam, USA, ab90529), Bcl-2 (1 : 1000, Abcam, USA, ab59348), Bax (1 : 1000, Abcam, USA, ab32503), cleaved caspase-9 (1 : 1000, Abcam, USA, ab2324), and cleaved caspase-3 (1 : 1000, Abcam, USA, ab2302) overnight at 4°C. After being washed with TBST for three times, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the enhanced chemiluminescence method was used to visualize the proteins.
5
Quantitative Protein Analysis by Western Blot
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A total of 4 × 105 cells were mixed with 1 ml RIPA solution (Sangon) to extract protein. Following denaturing in boiled water for 5 min, 10% SDS-PAGE gel was used to perform electrophoresis. PVDF membranes were used to perform gel transfer and blocking was performed for 2 h at room temperature in 5% non-fat milk. Blotting was performed using rabbit p-AKT (1:1200, ab18206, Abcam), AKT (1:1200, ab126811, Abcam), PI3K (1:1200, ab182651, Abcam), PI3K (1:1200, ab5451, Abcam), cytochrome c (1: 1200, ab90529, Abcam), and PI3K (1:1200, ab9485, Abcam) primary antibodies (overnight at 4°C) and goat IgG-HRP secondary antibody (1:800, MBS435036, MyBioSource). ECL (Sangon) was used to develop signal. Gray values were processed using Image J V1.6 software. Three independent replicates were set for each experiment.
6
Quantifying Mitophagy via Fluorescence Microscopy
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Cells were first fixed with 4% paraformaldehyde for 30 min at room temperature. After incubation with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, the samples were treated with the primary antibodies at 4 °C overnight. After the slides were washed with PBS and then incubated with secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min. Nuclei were stained using DAPI. Images were observed with fluorescence microscopy (Olympus BX-61). The primary antibodies used in the present study were as follows: p-ERK (1:1000, Abcam, #ab176660), Bnip3 (1:1000, Cell Signalling Technology, #44060), Tom20 (mitochondria marker, 1:1000, Abcam, #ab186735), LAMP1 (lysosome marker, 1:1000, Abcam, #ab24170), cyt-c (1:1000; Abcam; #ab90529). Mitophagy is the result of fusion between mitochondria and lysosome. The green mitochondria locate with red lysosome would generate the orange mitophagy. Then, the number of orange dot was measured to quantify the number of mitophagy.
7
Comprehensive Western Blot Analysis
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Standard Western blot analysis protocols were used. A 10% Input of IP lysates was loaded in each well as loading control. We used specific individual antibodies and dilutions (Parkin: abcam #ab15954 (1:1000), VCP: Invitrogen #MA3-004 (1:1000), FREMT3: abcam #ab68040 (1:1000), PDIA: abcam #ab2792 (1:1000), ILK: abcam #ab52480 (1:1000), 14-3-3: Thermo Fisher scientific #51-0700 (1:1000), prohibitin: abcam #ab28172 (1:1000), LAMP1: cell signaling #9091 (1:1000), HADHA: abcam #ab203114 (1:1000), PKM: abcam #ab137791 (1:1000), cofillin-1: GeneTex #GTX102156 (1:1000), Rac1: Sigma–Aldrich #SAB4502560 (1:1000), GAPDH: cell signaling #3683 (1:1000), Cytochrome C: abcam #ab90529 (1:2000), CD62P: abcam #182135 (1:1000), Actin: Santa Cruz #SC47778 (1:1000)).
8
Immunodetection of Inflammasome Components
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Rabbit monoclonal Anti-ASC (AG-25B-006-C100, AdipoGen, San Diego, CA, USA), mouse monoclonal anti-NLRP3 (AG-20B-0014-C100), rabbit monoclonal anti-caspase-1 (p20) (AG-20B-0042-C100), Rabbit monoclonal anti-GSDMD (ab209845, ABCAM, Cambridge, UK), rabbit monoclonal anti-PBR (ab109497, ABCAM), mouse Antiβ-actin (SJ190a9b68548, Sigma-Aldrich, St. Louis, MO, USA), rabbit monoclonal anti-IL-1β (12242, CST, Danvers, MA, USA), rabbit monoclonal anti-Iba1 (PA5-21274, Invitrogen), mouse monoclonal anti-Iba1 (019-19741, WAKO, Osaka Japan), anti-cytochrome C antibody (ab90529, Abcam) FITC anti-mouse CD45 antibody (103108, BioLegend, San Diego, CA, USA), APC anti-mouse F4/80 antibody (123116, BioLegend), PE/Cyanine7 anti-mouse CD86 antibody (105014, BioLegend), PE anti-mouse/human CD11b antibody (101208, BioLegend).
9
Western Blot Analysis of Apoptotic Markers
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SW480 cells were collected at the treatment time-points with reagents as suggested in each experiment. Cells were lysed with RIPA buffer (Thermo Fisher Scientific) and 15% electrophoresis (Thermo Fisher Scientific) was conducted under 90 V for 2 h. After undergoing electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. Primary antibodies, rabbit polyclonal anti-fibronectin antibody (1:500), rabbit polyclonal caspase-3 antibody (1:500, ab2302), rabbit polyclonal NF-κB antibody (1:500, ab7971), rabbit polyclonal p53 antibody (1:500, ab1431), rabbit polyclonal PARP (1:500, ab6079), rabbit polyclonal Bax antibody (1:500, ab53154), rabbit polyclonal cytochrome c antibody (1:500, ab90529) and rabbit polyclonal GAPDH antibody (1:500, ab9485) (all from Abcam) were incubated with the membranes for 24 h at 4°C. After washing with TBST buffer three times, the secondary antibody goat anti-rabbit IgG H&L (HRP) was incubated at room temperature for 1 h. Immunostaining was carried out using DAB Plus substrate and chemiluminescence system (Amersham Biosciences, Freiburg, Germany). The results were analyzed using chemiluminescence Molecular Imager® ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
10
Immunofluorescence Staining of Mitochondrial Proteins
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Cells were plated on glass slides in a 6-well plate at a density of 1 × 106 cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature [35 (link)]. The cells were then incubated with the primary antibodies: Tom20 (1:1000, Abcam, #ab186735), MIEF1 (1:1000, Abcam, #ab89944), p-JNK (1:1000; Cell Signaling Technology, #9251) and cyt-c (1:500; Abcam; #ab90529) [36 (link)].
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