Slices from frozen livers (10 μm thickness) were prepared and immunohistochemically stained as previously described (Braeuning et al, 2010 (link)), using antibodies against glutamine synthetase (GS; 1 : 1000, Sigma), β-catenin (1 : 50, Cell Signaling, Danvers, MA, USA) or BrdU (1 : 50, Dako, Glostrup, Denmark) in combination with horseradish peroxidase-conjugated secondary antibodies directed against rabbit (1 : 100, Dako) or mouse (1 : 20, Sigma) immunoglobulins with 3-amino-9 ethylcarbazole/H2O2 as substrates. For double staining of GS and Cx32, GS was visualised by the use of β-galactosidase-conjugated secondary antibodies (1 : 50, American Qualex, San Clemente, CA, USA) in combination with an antibody against Cx32 (1 : 250, Invitrogen/Zymed, Darmstadt, Germany) and horseradish peroxidase-conjugated secondary antibodies directed against rabbit (1 : 100, Dako) immunoglobulins with 3-amino-9-ethylcarbazole/H2O2 as substrates.
Horseradish peroxidase conjugated secondary antibody
Manufactured by Agilent Technologies
Sourced in Denmark, United States, United Kingdom, Germany, France, Belgium, Japan, Canada
Horseradish peroxidase-conjugated secondary antibodies are laboratory reagents used in immunoassays and other biochemical applications. They consist of secondary antibodies that are chemically conjugated to the enzyme horseradish peroxidase. The enzyme component can be used to catalyze a colorimetric or chemiluminescent reaction, enabling the detection and quantification of target proteins or analytes.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Chemidoc touch imaging system, by Bio-Rad (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Phosphatase inhibitor cocktails 1 and 2, by Merck Group (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (6 mentions)
Pierce ecl, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Enhanced chemiluminescence, by GE Healthcare (5 mentions)
Gapdh, by Abcam (5 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (5 mentions)
Image lab 5, by Bio-Rad (5 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (5 mentions)
Imagequant las 4000 mini, by GE Healthcare (4 mentions)
Ecl prime, by GE Healthcare (4 mentions)
Ecl system, by Thermo Fisher Scientific (4 mentions)
254 protocols using horseradish peroxidase conjugated secondary antibody
1
Immunostaining of Liver Sections
2
Apoptosis and Necroptosis Pathway Assays
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DHA (D2534) and EPA (E2011) were purchased from Sigma. DHA and EPA were dissolved in ethanol to produce a 100 mM stock solution. Annexin V apoptosis detection kit (88-8005-74) was obtained from Thermo Fisher. Bortezomib was obtained from LC laboratories and dissolved in DMSO. Pan-caspase inhibitor Z-VAD-FMK (tlrl-vad) was purchased from In vivoGen. Caspase-3 inhibitor Z-DEVD-FMK (FMK004), caspase-8 inhibitor Z-IETD-FMK (FMK007), caspase-10 inhibitor Z-AEVD-FMK (FMK009) and MG132 (1748) were obtained from R&D systems. Bis(sulfosuccinimidyl) suberate (BS3) (21586) were obtained from Thermo Scientific Pierce Biotechnology. Anti-Flag M2 Magnetic Beads was obtained from Sigma-Aldrich. Antibodies for MLKL (#14993), RIPK1 (#3493), RIPK3 (#13526), phospho-Ser358-MLKL (#91689), phospho-Ser166-RIPK1 (#65746), phospho-Ser227-RIPK3 (#93654), cleaved-caspase3 (#9661) and Flag M2 antibody (#2368) were purchased from Cell Signaling Technology; anti-GAPDH (sc-47724) was obtained from Santa Cruz Biotechnology. Anti-MLKL antibody (GTX107538) for immunoprecipitation was obtained from GeneTex (Hsinchu, Taiwan). Horse radish peroxidase-conjugated secondary antibodies were purchased from Dako Agilent.
3
Antibody-based Protein Detection Assay
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Antibodies used were goat anti-GST antibody (GE Healthcare, Piscataway, NJ), palladin (reacts with the 90-, 140-, and 200-kDa isoforms), or a 4Ig isoform-selective palladin (reacts with the 140- and 200-kDa isoforms; Ronty et al., 2006 (link)), HA and MT1-MMP rabbit polyclonal antibodies (Abcam, Cambridge, United Kingdom), and mouse monoclonal antibodies against MT1-MMP (antibody against the catalytic domain recognizing both the latent and active protease), cortactin, phospho-tyrosine (EMD Millipore Corporation, Temecula, CA), HA (Cell Signaling Technology, Danvers, MA), α-actinin, α-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich, St. Louis, MO), phospho-FAK, E-cadherin, N-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ), vimentin (Santa Cruz Biotechnology, Dallas, TX), and cadherin 11 (Invitrogen), as well as Alexa Fluor 647–phalloidin secondary antibodies (Invitrogen), and horseradish peroxidase–conjugated secondary antibodies (Dako, Agilent Technologies, Santa Clara, CA). We also used GM6001, and PP2 inhibitors (Millipore), PDGF-AB (R&D Systems, Minneapolis, MN), enhanced chemiluminescence (GE Healthcare), phalloidin–tetramethylrhodamine isothiocyanate, and collagen type I from rat tail (Sigma-Aldrich).
4
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer after which protein content was determined using a DC protein assay (Bio-rad) according to manufacturer’s instructions. Western blot analysis was performed as described before.22 (link) Briefly, equal amounts of protein were separated with electrophoresis using 10%–12% SDS-polyacrylamide gels under reducing conditions. Proteins were transferred to PVDF membranes (Sigma) and non-specific binding was blocked with 5% milk powder in tris-buffered saline containing 0.05% Tween-20 (Merck, Darmstadt, Germany). Blots were incubated overnight with mouse anti-CTSS (Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-TAP1 hybridoma supernatant (clone mAb 148.3, E. Wiertz, Dept. of Medical Microbiology, UMC Utrecht, The Netherlands), rabbit anti-V5 tag (Abcam, Cambridge, UK) or mouse anti-β-actin (Santa Cruz Biotechnology). Detection was performed by horseradish peroxidase-conjugated secondary antibodies (all from Agilent, CA, USA) and chemiluminescence (Thermo Fisher Scientific) was used to visualize the target proteins.
5
Western Blot Analysis of Caco-2 Cells
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Caco-2 cells were lysed in lysis buffer (CelLytic Mammalian Cells, Sigma Aldrich®, Saint-Quentin Fallavier, France) containing protease inhibitors (Complete, Roche®, Meylan, France). The protein concentration was measured using the BCA colorimetric method (BC Assay Protein Quantitation Small Kit, Interchim®, Montluçon, France) and, for each experimental condition, an equal amount of total proteins was separated on 8% SDS polyacrilamide gel and then transferred onto PVDF membranes. Blots were blocked overnight in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skimmed milk (Sigma Aldrich®, Saint-Quentin Fallavier, France). The PVDF membranes were further stained for 1 h at room temperature with a mouse monoclonal anti β-actin antibody (Ambion, Life technology®, Saint Aubin, France) or a mouse monoclonal anti-ZO-1 antibody (Invitrogen, Life Technology®, Saint Aubin, France). Blots were then incubated for 1h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Dako, Agilent Technology®, Les Ulis, France) and revealed by chemiluminescence (ECL plus, Amersham, GE Healthcare Life Science®, Velizy-Villacoublay, France) before printing on radiographic film (Hyperfilm, Amersham, GE Healthcare Life Science®, Velizy-Villacoublay, France).
6
Western Blot Analysis of PDE4B and PDE4D
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The protein lysates were resolved using sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Immobilon, Merck-Millipore; Burlington MA, USA, IPVH00010). The blots were blocked with 5% non-fat dry milk in Tris-buffered saline, 0.05% Tween 20 (TBST) buffer at room temperature for 1 h. The membranes were then incubated overnight with antibodies directed against PDE4B (NBP2-01171, Novus Biologicals, Minneapolis, MN, USA), PDE4D (ab171750, Abcam, Cambridge, UK) and ß-actin (A5441, Sigma-Aldrich). The appropriate horseradish peroxidase-conjugated secondary antibodies (Dako Products, Agilent, Santa Clara, CA, USA) and the LuminataTM Western horseradish peroxidase (HRP) Substrate (Immobilon, Merck-Millipore) were used to detect the bound antibodies. The size of the detected proteins was estimated using protein molecular-mass standards (Hyperpage Prestained Protein Marker; Bioline, Paris, France). ß-actin was used to verify the equal loading of the protein on each lane.
7
Western Blot Analysis of Protein Expression
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Tissue samples were homogenized in liquid nitrogen and dissolved in buffer containing 250 mM sucrose, 10 mM triethanolamine, protease inhibitor (cOmplete™; Roche) as previously described (Seidel et al., 2012 (link); Neymeyer et al., 2015 (link)). Samples were sonicated and nuclei removed by centrifugation (1,000 ×g for 10 min). Postnuclear supernatants were separated on 10% polyacrylamide mini-gels. Proteins were transferred to nitrocellulose membranes. Unspecific binding sites were blocked with 5% skim milk in PBS for 1 h at room temperature. Antibodies were diluted in milk as detailed in Supplementary Table S1 and applied overnight at 4°C. The abundance of α-tubulin was determined in parallel and served as loading control. Detection of bound primary antibodies was performed by incubation with horseradish peroxidase-conjugated secondary antibodies (Agilent Technologies, Santa Clara, United States) diluted in milk. Signals were generated by incubation with ECL solution (Western Blotting Detection Reagents, GE Healthcare). Image acquisition and signal quantification were performed using an Intas ECL ChemoCam Imager HR 3.2/6.0 (Intas Science Imaging Instruments, Göttingen, Germany).
8
Immunoblotting: Cellular Protein Quantification
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Lysates from the CEM or NALM6 cell lines were separated by electrophoresis on an SDS‐polyacrylamide gel. The proteins were then electroblotted onto a Hybond‐P PVDF membrane. Protein expression was analyzed using the primary antibodies anti‐GAPDH (Santa Cruz Biotechnology #sc‐20357) and anti‐NHP2L1 (Abcam #ab95958). Horseradish peroxidase–conjugated secondary antibodies were purchased from Agilent (Santa Clara). Phospho‐histone H3 (Ser10) clone 3H10 (Millipore #05‐806); Beta tubulin (Sigma‐Aldrich#C4585) Histone H3 (proteintech#17168‐1‐AP) were used. Sororin was provided by Dr. Jan‐Michael Peters from the Research Institute of Molecular Pathology, Vienna, Austria.
9
Western Blot Analysis of Protein Lysates
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To prepare the total cell lysates, cells were resuspended in SDS-lysis buffer and heated for 10 min at 98 ºC. The protein extracted was quantified with the BCA Protein Assay Kit. For Western blotting, cell lysates were resolved on SDS-polyacrylamide gels (SDS-PAGE) and transferred onto Hybond-ECL nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 10% non-fat milk diluted in TBS-T (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Tween-20) and then incubated for 16 h at 4 ºC with the primary antibodies diluted in TBS-T containing 5% non-fat milk (or bovine serum albumin [BSA] for the phosphospecific antibodies). After several washes in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Dako, Agilent Technologies, Santa Clara, California) for 1 h at room temperature diluted in TBS-T containing 5% non-fat milk. After washing in TBS-T, the chemiluminiscence signal was revealed with Western Lightning® Plus ECL and measured in a LAS-3000 image analyzer (Fuji PhotoFilm, Tokyo, Japan) with the LAS3000-Pro software. Band intensities were quantified with the ImageQuant TM TL software or ImageStudio TM Lite, and the relative protein levels were calculated using α-tubulin or vinculin as loading controls. Primary antibodies used are listed in Key Resources table.
10
Western Blot Analysis of Endothelin-1, NOX2, and OXPHOS
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Protein concentrations of the LV tissues were determined by the BCA protein assay kit (Thermo Fisher, Erembodegem, Belgium). Western blot was performed as previously described40 (link). Briefly, equal amounts of proteins (15 µg) were separated on a 12% SDS-PAGE gel with a mini protean 3 electrophoresis system (Bio-rad Laboratories, Temse, Belgium), transferred to a polyvinylidene fluoride (PVDF) membrane and subsequently, blocked for 2 h with 5% milk in Tris-buffered solution containing 0.1% Tween-20 (TBS-T) followed by incubation overnight at 4 °C in the presence of a specific endothelin-1 antibody (1/2500, Abcam, ab117757, Cambridge, United Kingdom), NOX2 antibody (1/2500, Abcam, ab31092, Cambridge, United Kingdom) or an OXPHOS antibody (1/1000, Abcam, ab110411, Cambridge, United Kingdom). Horseradish peroxidase-conjugated secondary antibodies (DAKO, Belgium) at a dilution of 1/2000 were used. Both primary and secondary antibodies were diluted in 5% milk-TBS-T. Visualization was performed with the enhanced chemiluminescence (ECL) technique using the Pierce ECL Plus western Blotting Substrate (Thermo Fisher, Erembodegem, Belgium). Data were normalized to β-actin protein levels.
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