FZU-0038-056 or DMSO treated cancer cells were harvested using lysis buffer supplemented with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) for 30 min on ice. Proteins were collected and centrifuged at 4 °C, 13000 rpm for 10 min. Equal amounts of protein samples were then separated by SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The membrane was blocked in 5 % skim milk for 1 hour, incubated with the primary antibody (1000 × dilution) overnight at 4 °C, washed 3 times with 1 × PBST for 10 min/time, and then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratory, West Grove, PA) for 1 hour. They were then washed 3 times again with 1 × PBST. Finally, the membranes were incubated with Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Shelton, CT) and images were taken using an ImageQuant LAS4000 Biomolecular imager (GE Healthcare, UK).
Horseradish peroxidase hrp
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase (HRP) is a commonly used enzyme in various laboratory applications. It catalyzes the oxidation of substrates in the presence of hydrogen peroxide, producing a colored or luminescent product. HRP is a widely used reporter molecule in immunoassays, Western blotting, and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Confocal laser scanning microscope, by Olympus (3 mentions)
Trans blot polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Anti sars cov 2 s rbd, by Sino Biological (2 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (2 mentions)
Anti vsv m 23h12, by Kerafast (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Sybr green realtime pcr master mix, by Toyobo (2 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Western lighting chemiluminescence reagent plus, by PerkinElmer (1 mentions)
Ab209845, by Abcam (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ag 20t 0138 c100, by Adipogen (1 mentions)
Luminata forte chemiluminescence substrate, by Merck Group (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ag 20b 0042 c100, by Adipogen (1 mentions)
40 protocols using horseradish peroxidase hrp
1
Western Blot Analysis of Protein Expression
2
Signaling Blot Protein Detection
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For signaling blots, the supernatant was removed, and cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors plus 4× Laemmli sample buffer. Caspase and GSDMD cleavage were measured from the combined cell lysate and supernatants. Proteins were separated via SDS-PAGE with 8 to 12% polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore), and blocked with 5% nonfat dry milk. Primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), caspase-3/cleaved-caspase-3 (9662 and 9661, CST), caspase-7/cleaved-caspase-7 (9492 and 9491, CST), caspase-8/cleaved-caspase-8 (4927, CST and AG-20T-0138-C100, Adipogen), caspase-9 (9504, CST), GSDMD (ab209845, Abcam), p-RIPK3 (91702, CST), RIPK3 (2283, ProSci), p-MLKL/MLKL (37333 and 37705, CST), and β-actin (4970, CST) were incubated overnight at 4°C followed by appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) incubated for 1 h at room temperature (Jackson ImmunoResearch, West Grove, PA). Membranes were visualized using Luminata Forte Chemiluminescence substrate (WBLUF0500, Millipore) or SuperSignal West Femto substrate (34096, Thermo Fisher Scientific) on a Bio-Rad ChemiDoc.
3
Western Blot Analysis of Viral Proteins
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Supernatant samples containing VSV were mixed 1:1 with sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis sample buffer containing 20% β-mercaptoethanol and heated to 99°C for 10 min. SDS-PAGE and transfer to Trans-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories) of all samples was performed as described elsewhere (34 (link)). Protein detection was performed using anti-SARS-CoV-2 S RBD (1:1000; Sino Biological) or anti-EBOV GP (ZGP 12/1.1, 1 μg/ml; kindly provided by Ayato Takada, Hokkaido University, Japan) or anti-VSV M (23H12, 1:1000; Kerafast Inc.). After horse-radish peroxidase (HRP)-labeled secondary antibody staining using either anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:5000) (Jackson ImmunoResearch), the blots were imaged using the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) and an iBright™ CL1500 Imaging System (Thermo Fisher Scientific).
4
Protein Extraction and Western Blot Analysis
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Brain regions (OE, OB, cerebral cortex, cerebellum) were homogenized by a motor-driven pestle on ice in 1× RIPA buffer (Millipore) supplemented with 1 mM phenylmethane sulfonyl fluoride (PMSF), 1 mM Na3VO2, 10 mM NaF, phosphatase inhibitor cocktails (Sigma), and Roche Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet (Roche). Homogenates were centrifuged at 10,000 × g for 10 min at 4°C; the supernatants were analyzed by performing Western blotting using 20 μg of total protein on Novex NuPAGE 4–12% Bis-Tris protein gels (Thermo Scientific). Membranes were washed with PBS with 0.05% Tween-20 (PBST), then blocked with 5% milk/PBST or 5% BSA/PBST for 1 hr at 21–23°C, followed by overnight incubation with primary antibodies at 4°C: anti-TNFα (1:250, mouse; R&D Systems), anti-3-nitrotyrosine (1:1,000, rabbit; Millipore), anti-4-hydroxynonenal (1:250, mouse; R&D Systems), anti-olfactory marker protein (OMP) (1:400, goat; Santa Cruz), anti-cleaved caspase 3 (1:1,000, rabbit; Cell Signaling), anti-caspase 3 (1:1,000, rabbit; Cell Signaling), anti-actin (1:10,000, mouse; Sigma). Horseradish peroxidase (HRP; 1:10,000, goat; Jackson)-enhanced chemiluminescence was detected using West Pico Chemiluminescent Substrate (Thermo Scientific). The density of the bands was assessed using ImageJ (Abràmoff et al. 2004 ).
5
Immunoblot Analysis of ApoM Fusion Proteins
6
Immunoblotting Analysis of IL-1β Signaling
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The expression of IL-1β in supernatants was analyzed using SDS-PAGE. After transfer onto PVDF membranes, non-specific antibody binding was blocked for 1 h at room temperature using 5% skim milk. Membranes were then incubated for 1 h at room temperature or 24 h at 4C with anti-human IL-1β antibody (R&D systems), anti-signal transducer and activator of transcription 3 (STAT3) antibody and anti-phospho-STAT3 antibody (Cell Signaling Technology, Inc., Boston, MA), anti-caspase-1 antibody (Cell Signaling) followed by incubation for 1 h with secondary antibody conjugated horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, Inc., PA). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (Takara Bio Inc., Shiga, Japan).
7
Drosophila Synapse Immunostaining and Imaging
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Third instar larvae were dissected in HL3 buffer and subsequently fixed in HL3 + 3.7%PFA for 20 min. Tissue was permeabilized using 1× PBS with 0.2% Triton-X and 5% BSA. Staining was performed using the following probes/antibodies: horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, 1:250), Disc Large 1 (4F3, DHSB, 1:50), mouse monoclonal YARS1 (Abnova, 1:500), rabbit polyclonal GFP (Invitrogen, 1:2000), rabbit polyclonal RFP (Abcam, 1:250), mouse monoclonal Brp (nc82, DHSB, 1:100), mouse monoclonal FasII (1D4, DHSB, 1:250), mouse monoclonal Synapsin (SynORF1, 3C11, DHSB, 1:500). Alexa Fluor®−488 and Alexa Fluor®−546 secondary antibodies were used (Invitrogen, 1:1000). Muscle 6/7 of abdominal hemisegments 3 and 4 were imaged.
Laser scanning confocal microscopy was performed on a Carl Zeiss LSM700 microscope equipped with a 20× Plan-Apochromat (0.8 NA) or 63× Plan-Apochromat (1.4 NA) objective. Super-resolution structured illumination microscopy was performed on a Zeiss ELYRA S.1 microscope equipped with a 63× Plan-Apochromat objective (1.4 NA). For a description of methods used to calculate Lifeact-RFP distribution at synaptic boutons, please see Supplementary Fig.7 .
Laser scanning confocal microscopy was performed on a Carl Zeiss LSM700 microscope equipped with a 20× Plan-Apochromat (0.8 NA) or 63× Plan-Apochromat (1.4 NA) objective. Super-resolution structured illumination microscopy was performed on a Zeiss ELYRA S.1 microscope equipped with a 63× Plan-Apochromat objective (1.4 NA). For a description of methods used to calculate Lifeact-RFP distribution at synaptic boutons, please see Supplementary Fig.
8
BDNF Protein Quantification in Opioid-Treated Mice
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VTA brain punches from opioid-naïve and -dependent mice treated with or without minocycline were assayed for BDNF protein content using western blot (Supplementary Material and Methods ). Equal amounts of protein (25 ug) were loaded onto pre-cast 10% polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes. Membranes were incubated with an antibody against BDNF (1:150; Millipore, Billerica, MA) and visualized with a goat anti-rabbit antibody conjugated to a horseradish peroxidase (HRP; Jackson Immunoresearch, West Grove, PA). Results were analyzed with a computer-assisted densitometry analysis (Image J Software, NIH).
9
Immunodetection of Tau Proteins in rTg4510 Mice
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Cell lysates and media containing scFvs and iBs were diluted 1:10 with PBS and applied to paraffin-fixed brain tissue slides from 10-month-old rTg4510 mice. Immunohistochemical staining was done with a biotinylated anti-c-myc secondary antibody. For direct ELISA, MaxiSorp plates (Thermo Scientific, Waltham, MA, USA) were coated with 1 μg/mL recombinant tau, or the same tau protein phosphorylated in vitro (p-tau), prepared as described previously75 (link) in PBS and blocked with 5% fetal bovine serum (FBS)/PBS. Cell lysates and media were applied to plates, chicken anti-c-myc secondary antibody was conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as detection, and absorbance was measured at 450 nm.
10
Immunofluorescence Staining Protocol for Hep3B and SNU449 Cells
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Hep3B and SNU449 cells were seeded in 24‐well plates at a density of 3 × 104 cells per well. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. After fixation, they were permeabilized with 0.5% Triton X‐100 for 15 min at 4°C. Following this, the cells were exposed to probes and incubated at 55°C for 4 h. After incubation, the cells were washed for 5 min with 2× PBS and then treated with secondary antibodies conjugated with horseradish peroxidase (HRP) obtained from Jackson, West Grove, PA, USA. Finally, the cells were counterstained with DAPI, and images were captured using an Olympus confocal laser scanning microscope.
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