Postmortem MFG tissues were homogenized in TBS buffer at a ratio of 1:10 (wt/vol) with Pierce Protease and Phosphatase Inhibitor Cocktail (A32965, ThermoScientific) on ice. Tissue lysate was sonicated and then centrifuged at maximum speed for 15 min at 4 °C. Protein concentrations were measured using the BCA protein assay kit (Bio-Rad Laboratories, Inc.). Electrophoresis was performed using 30 μg of protein lysates, resolved in a 4–12% SDS-PAGE gel (CriterionTM TGXTM, Bio-Rad Laboratories, Inc.) and transferred to a nitrocellulose membrane (Immobilon®-P, Millipore) that was blocked with 5% BSA in TBS with 0.01% tween, followed by overnight incubation of primary antibodies; HT7 (1:300, MN1000, Thermo Fisher), AT8 (1:1000, MN1020, Invitrogen) and PHF1 (1:1000, Peter Davies antibodies) diluted in the blocking solution. Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse HRP conjugated (1:10,000, 626820, Invitrogen) were incubated for 2 h at RT and the proteins were detected with Supersignal West Pico (34580, Thermo Scientific) and imaged by using iBright 1500 (Invitrogen). Western blots were analyzed using ImageJ Fiji.
Mn1000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MN1000 is a versatile laboratory instrument designed for the analysis and processing of samples. It features advanced technology and precise controls to deliver consistent and reliable results. The core function of the MN1000 is to provide users with a reliable and efficient tool for their laboratory operations.
Automatically generated - may contain errors
Lab products found in correlation
Mn1020, by Thermo Fisher Scientific (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Immobilon p, by Merck Group (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Pierce protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Criterion tgxtm, by Bio-Rad (1 mentions)
Ibright 1500, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Ix51 inverted fluorescent microscope, by Olympus (1 mentions)
Sc 51670, by Santa Cruz Biotechnology (1 mentions)
Ab5622, by Merck Group (1 mentions)
Ab5076, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Ab1218, by Abcam (1 mentions)
A10036, by Thermo Fisher Scientific (1 mentions)
B2261, by Merck Group (1 mentions)
Fl 1171, by Vector Laboratories (1 mentions)
Sc 21796, by Santa Cruz Biotechnology (1 mentions)
L2880, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
12 protocols using mn1000
1
Quantitative Proteomic Analysis of Alzheimer's Disease
2
Quantifying Neuronal Morphology in Primary Cultures
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Primary striatal cultures were prepared as described [10 (link)], fixed with 4% paraformaldehyde 48 hours after plating, and processed for immunostaining following our published protocol [14 (link)] using the following primary antibodies: Tau (1:500, MN1000, ThermoFisher Scientific), TUJ1 (1:250, sc-51670, Santa Cruz), and MAP2 (1:1,000; AB5622, Millipore). Cells were visualized under an Olympus IX51 inverted fluorescent microscope.
NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ was used to analyze fluorescence microscopy images of neurites and nuclei of cultured primary neurons. The plugin was used to count neuronal nuclei, and traces and measure neurite length as described [88 (link)]. Ten randomly selected images of each neuronal culture type were processed. DAPI staining was employed as a nuclear stain. The average length was obtained by dividing the total length of the traces by the number of nuclear counts.
NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ was used to analyze fluorescence microscopy images of neurites and nuclei of cultured primary neurons. The plugin was used to count neuronal nuclei, and traces and measure neurite length as described [88 (link)]. Ten randomly selected images of each neuronal culture type were processed. DAPI staining was employed as a nuclear stain. The average length was obtained by dividing the total length of the traces by the number of nuclear counts.
3
Tau P301L Mutation Overexpression in Mice
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The cDNA encoding full-length 441–amino acid human 4-repeat tau bearing a P301L mutation with fusion protein GFP or GFP expression plasmids were cloned into a rAAV9 vector, which had been described previously [16 (link)]. Phosphate buffer saline (PBS, Hyclone); lipopolysaccharides (LPS, L2880, Sigma-Aldrich); glucocorticoid (GC, H20051748, Sinopharm).
The primary antibodies used in the present study: GFP (1:500, mouse, ab1218, Abcam); HT7 (1:500, mouse, MN1000, Thermo Fisher); Tau13 (1:500, mouse, sc-21796, Santa Cruz); IBA1 (1:500, goat, ab5076, Abcam); CD68 (1:200, rabbit, A15037, Abclonal); GFAP (1:500, rabbit, ab7260, Abcam); Lectin (1:500, FL1171, Vector Labs); IgG (1:300, A10036, Invitrogen); Hoechst (1:1000, B2261, Sigma-Aldrich); Occludin (1:300, 27260-1-AP, Proteintech), ZO-1 (1:300, 21773-1-AP, Proteintech), and ICAM-1 (1:300, 16174-1-AP, Proteintech).
The primary antibodies used in the present study: GFP (1:500, mouse, ab1218, Abcam); HT7 (1:500, mouse, MN1000, Thermo Fisher); Tau13 (1:500, mouse, sc-21796, Santa Cruz); IBA1 (1:500, goat, ab5076, Abcam); CD68 (1:200, rabbit, A15037, Abclonal); GFAP (1:500, rabbit, ab7260, Abcam); Lectin (1:500, FL1171, Vector Labs); IgG (1:300, A10036, Invitrogen); Hoechst (1:1000, B2261, Sigma-Aldrich); Occludin (1:300, 27260-1-AP, Proteintech), ZO-1 (1:300, 21773-1-AP, Proteintech), and ICAM-1 (1:300, 16174-1-AP, Proteintech).
4
Western Blot Analysis of Phosphorylated Tau
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Total and insoluble fractions were separated on a reducing 4–12 % Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently transferred to nitrocellulose membrane. Phosphorylated tau was detected using AT8 (1:1,000, MN1020, Thermo Fisher Scientific, Waltham, MA, USA) and total tau levels using HT7 antibody (1:1,000, MN1000, Thermo Fisher Scientific, Waltham, MA, USA), and protein expression was normalized with internal control anti-actin (1:10,000, Sigma-Aldrich, St. Louis, MO, USA).
5
Studying Tau Pathology in Rodent Neurons
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Rodent cortico-hippocampal neurons were cultured on cover glass pre-coated with Poly-D-lysine at a density of 1 × 106 cells per milliliter and cultivated for 48 h. Neurons were treated with fluorescently labelled (Alexa Fluor 488 or Fluor 594) sarkosyl-insoluble AD tau (2p, 100 nM) only or in combination with control and DC8E8/AX004 antibody (1 μM) and cultivated for 20 h in conditioned Neurobasal media at 37 °C, 5% CO2. Next day neurons were washed with pre-warmed PBS and mild trypsin (0,06% trypsin-EDTA, 3 min), fixed with 4% PFA-PHEM, pH 6.9 (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, PFA) for 12 min. Neurons were permeabilized with 0.1% Triton X100 in PBS (PBS-T) and blocked with 5% BSA in TBS-T. The cells were then incubated with anti-heparan sulfate antibody (10E4, AMSBIO 1:100) for 1 h or, in a different experiment with HT7 antibody (epitope against human tau, MN1000, Thermo Fisher Scientific 1:500), washed and incubated with secondary antibody goat anti-mouse Alexa Fluor 488 (Invitrogen). The samples were mounted in FluoroshieldTM medium with DAPI (Sigma-Aldrich). Images were captured by LSM 710 confocal microscope (Zeiss, Jena, Germany).
6
Tau Immunoprecipitation and Mass Spectrometry
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Tau was immunoprecipitated from 1 mg of total protein extracted from iPSC-derived neurons (120 DIV) using a polyclonal anti-tau antibody (Dako Cytomation). Immunoprecipitated samples were analyzed by western blot using a monoclonal tau antibody (MN1000; Thermo Fisher Scientific) or stained with colloidal blue (Thermo Fisher Scientific). Bands that corresponded to tau by western blot analysis were excised from the colloidal blue SDS-PAGE. Peptide masses of digested protein samples were determined using a Bruker ultrafleXtreme Maldi mass spectrometer in reflectron mode and ms/ms fragmentation performed in LIFT mode. Data analysis was with FlexAnalysis, BioTools and ProteinScape software (Bruker). Database searches of the combined mass fingerprint-ms/ms data were performed using Mascot (http://www.matrixscience.com ).
7
Immunostaining of Microglia and Tau
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Microglia were first washed five times with PBS, fixed with 2% PFA for 10 min, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 20 min followed by blocking in 1.5% normal goat serum in PBS-T for 1 h, and incubated overnight at 4 °C with mouse anti-human tau antibody (HT7, ThermoFisher MN-1000, 1:1000) to detect rTg4510 lysate-containing human tau and/or rabbit anti-Iba1 (WAKO, 1:500) to label microglia. The next day, samples were rinsed and incubated with secondary antibodies of goat anti-mouse and goat anti-rabbit AlexaFluor 488 and 555, respectively (Invitrogen). Nuclei were counterstained with DAPI. Images were captured with an EVOS cell imaging system (Thermo Fisher Scientific) or a Zeiss Axio Imager Z epifluorescence microscope (Zeiss) with a × 20 objective.
8
Tau and Neuroinflammation Protein Markers
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To examine full length tau and phosphorylated tau expression levels the following antibodies were used: HT7, which recognizes all forms of Tau protein, (1:40,000, MN1000, Thermo-Fisher Scientific, Waltham, MA, USA); AT8, which recognizes tau phosphorylated at residues Serine 202 and Threonine 205 (1:500, MN1020, Thermo-Fisher Scientific, Waltham, MA USA) and pTau (S202) antibody (1:5000, ab108387, abcam, Cambridge, MA USA), which recognizes only the phosphorylated residue at Serine 202. To examine for possible diffuse axonal injury (DAI) we measured levels of amyloid precursor protein, APP-A4, (1:2000, MAB348, Millipore-Sigma, Burlington, MA, USA). To determine effects on proteins known to be associated with neuroinflammation, microglial activation, and demyelination, we measured levels of soluble glial fibrillary acidic protein (GFAP), a marker of astroglial cells (1:20,000, NCL-L-GFAP-GA5, Leica Biosystems, Newcastle Upon Tyne, UK); ionized calcium-binding adapter molecule 1 (IBA-1), a marker for microglial cells, (1:1000, ab178847, Abcam, Cambridge, MA, USA); and myelin basic protein (MBP), a marker of myelination, (1:10,000, ab7349, Abcam, Cambridge, MA, USA).
9
Comprehensive Antibody Characterization Protocol
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Monoclonal antibodies used included: AT270 (MN1050, Thermo Fisher Scientific, RRID: AB_223651), AT180 (MN1040, Thermo Fisher Scientific, RRID: AB_223649), HT7 (MN1000, Thermo Fisher Scientific, RRID: AB_2314654), Tau5 (AHB0042, Thermo Fisher Scientific, RRID: AB_2536235), MAb359 (from Dr. Li Gan), anti-GAPDH (MAB374, Sigma-Aldrich, RRID: AB_2107445), anti-FLAG (F1804, Sigma-Aldrich, RRID: AB_262044), AT8 (MN1020, Thermo Fisher Scientific, RRID: AB223647), anti-SV2 (University of Iowa DSHB, RRID: AB_2315387), anti-PICK1 (75–040, Antibodies Inc, RRID: AB_2164544), anti-KIBRA (sc-133374, Santa Cruz Biotechnology, RRID:AB_2216359). Polyclonal antibodies used included: anti-PKMζ (from Dr. Todd C. Sacktor), anti-PSD95 (2507, Cell Signaling Technology, RRID: AB_561221), anti-HA (H6908, Sigma-Aldrich, AB_260070), GFP (A-21311, Thermo Fisher Scientific, RRID: AB_221477), anti-GluA1 (ABN241, Millipore, RRID: AB_2721164)
10
Visualization of Tau Filaments by TEM
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In vitro samples were negatively stained and studied by transmission electron microscopy (TEM). 4 μL of sample were adsorbed for 60 seconds on glow‐discharged copper grids coated with 2% parlodion and a continuous carbon film. Samples were stained with 2% uranyl acetate solution.
Labelling of tau filaments with immuno‐gold was adapted from Goedert et al.65 Samples were adsorbed on TEM grids as described above. Grids were laid face down on a drop of 0.1% gelatin (G7041, Merck KGaA) in DPBS, incubated for 10 minutes, transferred to a droplet of HT7 anti‐tau antibody (MN1000, Thermo Fisher Scientific, 1:20 dilution in DPBS), and incubated for 60 minutes. After washing in gelatin solution, grids were stained with anti‐mouse antibody conjugated to 10 nm gold beads (EM.GAF10). Uranyl acetate was used for negative stains.
Samples were imaged with FEI Tecnai T12 (operated at 120 kV) and FEI Tecnai G2 Spirit (operated at 80 kV) transmission electron microscopes (Thermo Fisher Scientific) equipped with TVIPS TemCam‐F416 (Tietz Video and Image Processing Systems GmbH) and EMSIS VELETA (EMSIS GmbH) cameras, respectively.
Measurements on the electron micrographs were done with the imageJ distribution Fiji (https://fiji.sc/ ). For Sup35NM seeded 2N4R wild‐type tau, periodicity of the wave pattern was used to calculate mean and standard deviation.
Labelling of tau filaments with immuno‐gold was adapted from Goedert et al.
Samples were imaged with FEI Tecnai T12 (operated at 120 kV) and FEI Tecnai G2 Spirit (operated at 80 kV) transmission electron microscopes (Thermo Fisher Scientific) equipped with TVIPS TemCam‐F416 (Tietz Video and Image Processing Systems GmbH) and EMSIS VELETA (EMSIS GmbH) cameras, respectively.
Measurements on the electron micrographs were done with the imageJ distribution Fiji (
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