Double DiD-labeled and luciferase-expressing PC-3 cells were injected into the left cardiac ventricle of the mice as described above. Mice were sacrificed on day 2 and 80 after injection, respectively, to detect the presence of double luciferase+ and DiD+ tumor cells in both tibiae of each mouse. Dissected tibiae were fixed with 4% paraformaldehyde and embedded in optimal cutting temperature (OCT) embedding compound (Sakura Finetek), and then frozen at −30°C before use. Serial 10 tissue sections (thickness: 10–12 µm) of both tibiae of all mice were longitudinally generated from a sagittal section with a Leica Cryostat CM3050 S. The tissue sections were stained with a rabbit anti-luciferase polyclonal antibody (L0159; Sigma) as described above. The whole fields of each slide were analyzed to detect double luciferase+ and DiD+ cells. Tissues were imaged on a Zeiss LSM 710 confocal microscope using either a 1.1 numerical aperture 40× water-immersion objective or a 1.4 numerical aperture 63× oil-immersion objective. The total number of double luciferase+ and DiD+ cells in each mouse was scored given by the two independent investigators and were statistically analyzed for further comparative number of luciferase+ and DiD+ cells in the indicated mice groups.
L0159
Manufactured by Merck Group
The L0159 is a laboratory equipment product offered by Merck Group. It is designed for general laboratory applications, but its core function is to provide a precise and reliable platform for conducting various experiments and analyses. The product specifications and technical details are maintained by the Merck Group, and no further interpretation or extrapolation on the intended use is provided.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Ab6556, by Abcam (2 mentions)
Anti ha hrp, by Merck Group (2 mentions)
Anti gal4 ad, by Takara Bio (2 mentions)
Anti his6 hrp, by Roche (2 mentions)
Polyclonal goat anti mouse hrp, by Bio-Rad (2 mentions)
Anti ack, by Cell Signaling Technology (2 mentions)
Ac k 103, by Cell Signaling Technology (2 mentions)
Precast sds page gel, by Bio-Rad (2 mentions)
Anti gfp, by Merck Group (2 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Oct embedding compound, by Sakura Finetek (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Cytoseal, by Thermo Fisher Scientific (1 mentions)
M4439, by Merck Group (1 mentions)
8 protocols using l0159
1
Quantification of Metastatic Tumor Cells
2
Chaperone-mediated protein cross-linking
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For the cross-linking reactions, 2 μM luciferase or LDH were incubated at 4 °C for controls or heat shocked at 45 °C or 55 °C, respectively, in the absence or presence of HSPB1-3D or HSPB1-3D-GxG in reaction buffer. The aggregates were then incubated at room temperature for 5 min with varying concentrations of disuccinimidyl suberate (DSS). Reactions were stopped with 30 mM Tris–HCl pH 7.5 and analyzed by immunoblots with antibodies specific for luciferase (L0159, Sigma), LDH (168–10,648, RayBiotech) and HSPB1 (5D12-A12, StressMarq Biosciences). Chemiluminescence detection was with ECL reagent (GE Healthcare), images were acquired in a ChemiDoc MP imaging system (Bio-Rad) and quantified using the Image Lab software (Bio-Rad) and ImageJ 1.46r (NIH).
3
Plant Protein Extraction and Analysis
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Plant tissues were collected into 2-ml tubes with metal beads and frozen in liquid nitrogen. After grinding with a tissue lyser (Qiagen, Germany) for 1 min at 30 rpm/s, proteins were extracted using protein extraction buffer (100 mM Tris–HCl [pH 8], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 2 mM dithiothreitol, 1× plant protease inhibitor cocktail, 1% NP-40, 2 mM phenylmethylsulfonyl fluoride, 10 mM Na2MoO4, 10 mM NaF, 2 mM Na3VO4) and incubating for 5 min. After centrifugation, the supernatants were mixed with SDS loading buffer, incubated at 70°C for 10 min, and resolved using SDS–PAGE. Proteins were transferred to a polyvinylidene fluoride membrane and monitored by western blot using anti-GFP (Abicode, M0802-3a) and anti-luciferase (Sigma, L0159) antibodies.
4
Bimolecular Luciferase Assay for CBL-CIPK Interactions
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LCI assays were performed using a previously described protocol (Zhou et al, 2018 (link)). The full‐length ORFs of OsCBL7 or OsCBL8 and OsCIPK7 were cloned into pCAMBIA1300‐cluc and pCAMBIA1300‐nluc respectively. CBL7‐cluc/OsCBL8‐cluc, OsCIPK7‐nluc plus N1‐54: mVenus: OsCRT3 or GFP were co‐expressed in N. benthamiana epidermal leaves for 3 days. The luminescence images were captured using a CCD imaging system. The relative luciferase activity was measured and analyzed by Image J software. Western blot was conducted using anti‐luciferase antibody (Sigma, L0159), anti‐cluc antibody (sigma, L2164), and anti‐GFP antibody (abcam, ab6556), respectively.
5
Histological Analysis of Brain Structure Changes
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Animals were euthanized 30 days after surgery. Animals were anesthetized with a cocktail of 120 mg/kg of ketamine and 10 mg/kg of xylazine before transcardial perfusion with phosphate-buffered saline, followed by 4% paraformaldehyde (PFA). After brain extraction, brains were postfixed in 4% PFA overnight at 4°C and then paraffin-wax embedded. Paraffin blocks were then sectioned coronally at 5-μm thickness and mounted on glass slides. Hematoxylin and eosin (H&E) staining was performed to analyze anatomical structure changes. Dako Autostainer (Universal Staining Systems, Carpinteria, CA) was used for immunohistochemistry, using the Dako EnVision+ System-HRP (DAB). Sections were deparaffinized with xylene and rehydrated through graded alcohols. Antigen retrieval was performed by steaming in distilled water for 30 min, and sections were then immunostained with antibodies against firefly luciferase (L0159; Sigma-Aldrich), glial fibrillary acidic protein (GFAP; PU020-UP; BioGenex, San Ramon, CA) and ionized calcium-binding adapter molecule 1 (Iba1; 019-19741; Wako, Richmond, VA). After chromogen development, slides were counterstained with Shandon Gill I Hematoxylin (ThermoFisherScientific, Waltham, MA) to visual nuclei. Slides were then dehydrated though graded alcohols and cleared with xylene before mounting coverslips with Cytoseal (ThermoFisherScientific).
6
Protein Extraction and Immunoblotting Protocol
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Protein extraction and immunoblotting were performed as described previously (Kim et al., 2003 (link)). Briefly, ground tissues were homogenized with extraction buffer (50 mM Tris–Cl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 3 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml antipain, 5 μg/ml chymostatin, 50 μM MG132, 50 μM MG115, and 50 μM ALLN) and centrifuged at 16,000 g at 4°C for 10 min. To detect luciferase protein, supernatant proteins were concentrated by TCA precipitation and resultant pellets were resuspended in Urea/SDS loading buffer (40 mM Tris–Cl, pH 6.8, 8 M Urea, 5% SDS, 1 mM EDTA, 2% 2-mercaptoethanol). The total proteins were separated on a 8% SDS-PAGE gel (acrylamide:bisacrylamide, 37.5:1) and probed with 1:1,000 anti-luciferase (Sigma, L0159) and 1:15,000 anti-ADK antibody (gift from Dr. David Bisaro), as a loading control. For immunoprecipitated proteins, 1:2,000 anti-myc (Sigma, M4439), 1:1,000 anti-HA (Sigma, 3F10), and 1:5,000 anti-GFP (Abcam, ab6556) were used.
7
Western Blot Protein Detection Protocol
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N. benthamiana leaf discs were ground in liquid nitrogen and proteins were extracted in Laemmli buffer 2X. Yeast protein were extracted after a lysis step in 0.1 M NaOH during 20 min at room temperature followed by protein extraction in Laemmli buffer 1X. Immunodetection of proteins were performed by loading the samples on precast SDS-PAGE gels (4-15%, Bio-Rad). Proteins were transferred on nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The nitrocellulose membranes were blocked with 1X Tris-buffered Saline with Tween20 (TBS-T) solution (137 mM NaCl, 0.1% Tween-20, 3% Milk). Proteins transferred on nitrocellulose membranes were stained in Ponceau S staining solution (0.5% Ponceau S (w/v), 1% acetic acid). Immuno-detection of the protein of interest were performed with the following antibodies : anti-GFP (1:3000, mouse mAb (clones 7.1 and 13.1), Sigma), anti-HA-HRP (1:5000, Rat mAb (clone 3F10), Sigma), anti-Gal4-AD (1:5000, mouse mAb, Takara), Anti-Ac-K (1:2000, mouse mAb Ac-K-103, Cell signaling), anti-His6-HRP (1:50000, mouse mAb (clone His-2), Roche), anti-luciferase (1:10000, L0159, Sigma), anti-rabbit IgG-HRP (1:10000, Cell signaling) and polyclonal goat anti-mouse-Hrp (1:10000, Bio-Rad).
8
Western Blot Protein Detection Protocol
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N. benthamiana leaf discs were ground in liquid nitrogen and proteins were extracted in Laemmli buffer 2X. Yeast protein were extracted after a lysis step in 0.1 M NaOH during 20 min at room temperature followed by protein extraction in Laemmli buffer 1X. Immunodetection of proteins were performed by loading the samples on precast SDS-PAGE gels (4-15%, Bio-Rad). Proteins were transferred on nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The nitrocellulose membranes were blocked with 1X Tris-buffered Saline with Tween20 (TBS-T) solution (137 mM NaCl, 0.1% Tween-20, 3% Milk). Proteins transferred on nitrocellulose membranes were stained in Ponceau S staining solution (0.5% Ponceau S (w/v), 1% acetic acid). Immuno-detection of the protein of interest were performed with the following antibodies : anti-GFP (1:3000, mouse mAb (clones 7.1 and 13.1), Sigma), anti-HA-HRP (1:5000, Rat mAb (clone 3F10), Sigma), anti-Gal4-AD (1:5000, mouse mAb, Takara), Anti-Ac-K (1:2000, mouse mAb Ac-K-103, Cell signaling), anti-His6-HRP (1:50000, mouse mAb (clone His-2), Roche), anti-luciferase (1:10000, L0159, Sigma), anti-rabbit IgG-HRP (1:10000, Cell signaling) and polyclonal goat anti-mouse-Hrp (1:10000, Bio-Rad).
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