For immunohistochemical labelling, tibial sections were first incubated with 0.1 % trypsin in PBS for 30 min at 37 °C, then washed in 0.025 % Triton in PBS (wash buffer). Endogenous peroxidase activity was blocked using 3 % H2O2 in methanol (30 min) followed by rinsing with wash buffer. The sections were blocked with 3 % BSA in 20 % rabbit serum and then incubated overnight at 4 °C with goat polyclonal anti-mouse sclerostin antibody (10 μg/ml) (AF1589, R&D Systems) and polyclonal goat IgG for control sections (AB-108-C, R&D Systems). Sections were washed and incubated for 30 min in horseradish peroxidase (HRP)-conjugated secondary antibody (rabbit anti-goat, 1:200) (P0160, Dako). They were then rinsed and HRP-label was visualised by incubating the sections for 5 min with 3,3′ diaminobenzidine (DAB)-solution. Three sections for each bone were counterstained briefly with haematoxylin and imaged using light microscopy to visualise osteocytes within cortical and trabecular bone. All sclerostin-positive osteocytes in bones of ZDF and lean rats were manually counted in these regions using image J.
P0160
Manufactured by Agilent Technologies
Sourced in United States
The P0160 is a laboratory equipment product offered by Agilent Technologies. It serves as a core function for performing various analytical tasks in a laboratory setting. The detailed specifications and intended use of this product are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
P0260, by Agilent Technologies (4 mentions)
P0448, by Agilent Technologies (3 mentions)
P0161, by Agilent Technologies (2 mentions)
Af1589, by R&D Systems (1 mentions)
Ab 108 c, by R&D Systems (1 mentions)
Ab8580, by Abcam (1 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
P0217, by Agilent Technologies (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ultra sensitive enhanced chemiluminescent, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Protease, by Roche (1 mentions)
Ab126595, by Abcam (1 mentions)
Ab256494, by Abcam (1 mentions)
Ab69090, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab125066, by Abcam (1 mentions)
Nb100 1798, by Novus Biologicals (1 mentions)
Nb300 318, by Novus Biologicals (1 mentions)
Bs 6313r, by Bioss Antibodies (1 mentions)
10 protocols using p0160
1
Immunohistochemical Quantification of Sclerostin
2
Antibody Labeling and Detection
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The primary antibodies are shown in Table 2 . The secondary antibodies for Western blot studies were horseradish peroxidase–conjugated anti-rabbit, anti-mouse or anti-goat IgGs (P0448 and P0161 or P0160, DAKO) and for immunofluorescence were anti-mouse, anti-rabbit or anti-guinea pig IgGs alexa-488, -555 or -647 labeled (Molecular Probes, Millipore).
3
Quantifying Polyglutamine Protein Levels
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The relative expression levels of some BlaP-polyQ chimeras and of Q82::YFP animals were confirmed by dot blot. For this purpose, worm extracts were prepared and collected as described before (Western blot analysis).
2 μl of each sample was spotted on a nitrocellulose membrane and the samples were allowed to dry for 1 h. The membrane was then incubated at room temperature (1) for 2 h in 5% blocking agent (GE Healthcare) and (2) with primary mouse anti-polyQ antibody (1/1000 dilution in Tris-Saline pH 7.6, 5TF1-1C2, Millipore) or primary rabbit anti-Histone H3 antibody (1/20000 dilution in Tris-Saline pH 7.6, ab8580, Abcam). Horse Radish Peroxidase-conjugated rabbit anti-mouse (1/50000 dilution in Tris-Saline pH 7.6, P0161, Dako) or goat anti-rabbit (1/50000 dilution in Tris Saline, pH 7.6, P0160, Dako) were used as secondary antibodies for visualization with Supersignal West Dura (Thermo Scientific).
Dot blot signals were analysed using ImageJ. Upon removal of background signals, the polyQ signal was normalized to the Histone H3 signal, which functioned as an endogenous loading control.
2 μl of each sample was spotted on a nitrocellulose membrane and the samples were allowed to dry for 1 h. The membrane was then incubated at room temperature (1) for 2 h in 5% blocking agent (GE Healthcare) and (2) with primary mouse anti-polyQ antibody (1/1000 dilution in Tris-Saline pH 7.6, 5TF1-1C2, Millipore) or primary rabbit anti-Histone H3 antibody (1/20000 dilution in Tris-Saline pH 7.6, ab8580, Abcam). Horse Radish Peroxidase-conjugated rabbit anti-mouse (1/50000 dilution in Tris-Saline pH 7.6, P0161, Dako) or goat anti-rabbit (1/50000 dilution in Tris Saline, pH 7.6, P0160, Dako) were used as secondary antibodies for visualization with Supersignal West Dura (Thermo Scientific).
Dot blot signals were analysed using ImageJ. Upon removal of background signals, the polyQ signal was normalized to the Histone H3 signal, which functioned as an endogenous loading control.
4
LHCGR Protein Purification and Western Blotting
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Protein purification and Western blotting (WB) was performed as described previously [17 (link)]. In brief, protein was purified from snap-frozen samples and cell pellets by homogenization in a lysis buffer supplemented with protease (Roche, Basel, Switzerland) and phosphatase inhibitors (Thermofisher scientific, Waltham, USA). 15 μg of protein was loaded in each lane. Primary antibodies were: LHCGR (c-terminal targeting, Aviva, # OASG04237), LHCGR (n-terminal targeting, Promab # Pro-30751, Ramlösa, Sweden) and β-actin (loading control, Santa Cruz, # sc-47778, Dallas, USA) all in a concentration of 1:200. Anti-rabbit (Dako, # P0217, Glostrup, Denmark), anti-goat (Dako, # P0160) and anti-mouse (Dako, # P0260) secondary antibodies were horseradish peroxidase (HRP) conjugated and used in a 1:500 dilution followed by development using ultra-sensitive enhanced chemiluminescent (Thermofisher scientific). Pictures were taken using ChemiDoc™ MP imaging system (Bio-rad, Hercules, USA) with automatic exposing time for intense bands. Experiments were repeated at least three times in independent experiments.
5
Comprehensive Antibody Panel for Oxidative Stress Analysis
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The following primary antibodies were used for immunoblot analysis and immunohistochemistry: mouse monoclonal anti–transferrin receptor antibody (13‐6800, Invitrogen), goat polyclonal anti–Lipocalin‐2/NGAL antibody (AF1857, R&D systems), rabbit monoclonal anti–β‐actin (ACTB) antibody (AC026, ABclonal), rabbit monoclonal anti–IRP1 antibody (ab126595, Abcam), rabbit polyclonal anti–IRP2 antibody (NB100‐1798, Novus), rabbit polyclonal anti–FTH1 antibody (3998, CST), rabbit polyclonal anti–ferritin light chain (FTL) antibody (ab69090, Abcam), rabbit polyclonal anti–divalent metal transporter 1 (DMT1) antibody (20507‐1‐AP, proteintech), rabbit polyclonal anti–8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) antibody (bs1278R, Bioss), rabbit polyclonal anti–4‐Hydroxynonenal antibody (bs6313R, Bioss), anti–CD10 antibody (ab256494, Abcam), anti–Ki67 antibody (ab16667, Abcam), rabbit monoclonal anti–Cleaved Caspase‐3 antibody (9664, CST), rabbit monoclonal anti–Glutathione Peroxidase 4 antibody (ab125066, Abcam), and anti–xCT antibody (NB300‐318, Novus). Secondary antibodies used in immunoblot analysis and immunohistochemistry were as follows: HRP‐conjugated polyclonal Goat anti–Rabbit antibody (P0448, DAKO), HRP‐conjugated polyclonal Rabbit anti–mouse antibody (P0260, DAKO) and HRP‐conjugated polyclonal Rabbit anti–Goat antibody (P0160, DAKO).
6
Western Blot Protein Analysis
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Samples from cell lines were scraped on ice with RIPA cell lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Roche). Proteins were separated by a Ready Gel precast gel (Bio-Rad) and transferred onto a nitrocellulose membrane. Blocking was done with 5% milk, 1 h at RT. Primary antibody incubations were performed overnight at 4° for both polyclonal NEUROD1 (1:500, PA5-47381; Invitrogen) and monoclonal β-tubulin (1:20,000, SAP.4G5). Secondary antibodies (polyclonal rabbit anti-mouse immunoglobulins/HRP [1:1,000, P0260; Dako] and polyclonal rabbit anti-goat immunoglobulins/HRP [1:2,000, P0160; Dako]) were incubated for 1 h at RT. All antibody dilutions were made to 5% milk. Proteins were detected by enhanced chemiluminescence (Bio-Rad).
7
Western Blot Analysis of Extracellular Vesicles
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Samples were mixed with 4× Pierce™ LDS Sample Buffer (#84788, ThermoFisher Scientific, Waltham, MA, USA) including DTT (0.3M) and denatured for 5 min at 75 °C. For Western blot analysis, 5 μL (fractions 6–8 in UF+SEC) or 25 μL (other samples) of protein sample was separated using a 4–20% SDS-PAGE (Mini-PROTEAN® TGX Stain-Free™ Protein gels, #4568094, BIO-RAD, Hercules, CA, USA) and MOPS running buffer and transferred to nitrocellulose membranes using a BIORAD Trans-Blot SD semi dry transfer system. Membranes were blocked for 45 min (PBS, 0.1% Tween 20, 5% dried milk) and primary antibodies against Tsg101 (ab83, Abcam, Cambridge, UK), Alix (ab117600, Abcam, Cambridge, UK), Tamm-Horsfall protein (uromodulin, ab733, Millipore, Burlington, MA, USA) and CD9 (#MA1-80307, Invitrogen, Carlsbad, CA, USA) were used at 1:1000 dilution o/n at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit anti-goat (#P0160, Dako, Agilent Technologies, Santa Clara, CA, USA, 1:5000) and rabbit anti-mouse (#P0260, Dako, 1:5000)) were used and chemiluminescence activated using the Amersham ECL Prime detection reagent kit (#RPN2232 GE Healthcare, Chicago, IL, USA). Bands were visualised using the signal accumulation mode with a Bio-Rad ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA).
8
Antibodies for Protein Detection
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The following antibodies were used: rabbit anti-STAG1 (GTX129912; Genetex), goat anti-STAG2 (A300-158A; Bethyl Laboratories), mouse anti-β-actin (AC-15) (A3854; Sigma-Aldrich), mouse anti-GAPDH (ab8245; Abcam, Fig 2 ), rabbit anti-GAPDH (ab9485; Abcam, Fig 4 ), rabbit anti-H3pS10 (06570; Millipore), FITC-conjugated mouse anti-tubulin (F2168; Sigma-Aldrich), rabbit anti-SMC1 (A300-055A; Bethyl Laboratories), mouse anti-SMC3 (ID 646 in HEK293 co-immunoprecipitation immunoblot; Peters Laboratory), rabbit anti-SMC3 (A300-060A in capillary immunodetection assay; Bethyl Laboratories), mouse anti-RAD21 (05-908; Millipore), mouse anti-tubulin (T5168; Sigma-Aldrich), mouse anti-FLAG (1042E; Sigma-Aldrich, Fig S5 ), rabbit anti-FLAG (F7425; Sigma-Aldrich, Fig 4 ), rabbit anti-histone H3 (4499; Cell Signaling), mouse anti-p53 (OP43; Calbiochem), and secondary rabbit (1706515; Bio-Rad), mouse (1706516; Bio-Rad), and goat (P0160; Dako) anti-IgG-HRP.
9
Protein Extraction and Western Blot Analysis
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For western blot analysis, larvae/adult organs were taken and incubated for 10 min with 2 mm Natrium‐Vanadate in 1× PBS on ice to inhibit phosphatases and then lysed in NP40 lysis buffer (150 mmol L−1 NaCl, 50 mmol L−1 Tris‐HCl, pH 7.4, 1% NP40, 10 mmol L−1 EDTA, 10% glycerol, and protease inhibitors), followed by homogenization with TissueLyser II (Qiagen) and incubation on ice for 30 min on a shaker. The supernatant containing the protein lysate was diluted 5:1 with Laemmli sample buffer and boiled at 95 °C for 5 min, separated via SDS‐PAGE, and then transferred to a nitrocellulose membrane for antibody incubation (anti‐Akr 1a1a antibody 1:1000, anti‐Actin antibody A2228 from Sigma‐Aldrich, 1:1000, AKT antibody 9272S from CST, 1:1000, P‐AKT antibody 4060P from CST, 1:1000), secondary HRP‐conjugated antibodies 1:1000 (for β‐actin: rabbit anti‐goat, P0160, Dako; for Akr1a1a: goat anti‐guinea pig, ABIN101281, antibodies‐online.com, for P‐AKT and AKT, Goat anti‐Rabbit, P0448, Dako). Visualization by enhanced chemiluminescence (ECL) was acquired after incubation with HRP (Horseradish Peroxidase) substrate.
10
Adenoviral Transduction of HEK293 Cells
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HEK293 cells were infected with a multiplicity of infection (MOI) of 2 or 5 for 24–48 h with Ad5.CMV.sIL21R and Ad5.CMV.Null or Ad5.CMV.eGFP as negative controls. For immunocytochemistry, anti-IL21R antibody (AF596, R&D Systems, Minneapolis, MN, USA) and Alexa Fluor 488 donkey anti-goat secondary antibody (A11055, Invitrogen, Carlsbad, CA, USA) were used at a dilution of 1:100 and 1:200, respectively. For immunodetection, cell pellet was homogenized in lysis buffer (RIPA) and total protein was determined from lysed cells and supernatants using the Pierce BCA Protein Assay (232,227, Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts and supernatants were loaded onto denaturing acrylamide gels and then electrotransferred to PDVF membranes (10,600,023, GE Healthcare, Chicago, IL, USA). Anti-IL21R primary antibody (AF596, R&D Systems) was used at 1:1000 dilution and goat HRP-anti-IgG secondary antibody (P0160, Dako, Agilent Technologies, Santa Clara, CA, USA) at 1:10,000 were used in the presence of 5% (w/v) of BSA (A9418, Sigma-Aldrich, St. Louis, MO, USA).
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