Mice were deeply anesthetized using a CO2 before being euthanized by rapid decapitation. The brain was removed quickly, and different parts of the brain were rapidly dissected out on ice. The tissues were suspended in RIPA buffer and mechanically homogenized by using a polytron tissue homogenizer. Protein quantitation was performed using a BCA kit, and 20 μg of total protein was loaded for SDS-PAGE as performed previously [32 (link)]. Antibodies include anti-NIPP1 (1:1000; Sigma), anti-tubulin (1:2000; Chemi-con), anti-actin (1:4000; Sigma), anti-phospho-tau (S202, T205) (AT8) (1:500; Invitrogen), anti-p-tauThr231 (1:1000; Millipore), anti-tau (1:1000; Sigma), anti-PP1 pT320 (1:1000; Cell Signaling Technology), anti-PP1 (1:1000; E-9, Santa Cruz Biotechnology, Inc.), anti-pAKT (Ser473) (1:500; Cell Signaling), anti-AKT (1:1000; Cell Signaling), anti-pGSK3β (Ser9) (1:500; Cell Signaling), anti-GSK3β (1:1000; SCBT), anti-pERK1/2 (1:500; Cell Signaling), anti-pERK1/2 (1:1000; Cell Signaling), and anti-MBP (1:1000; Bio-Rad). Blots were quantified using AzureSpot. For p-tau epitopes, the more prominent top band was used for analysis.
Anti tau
Manufactured by Merck Group
Sourced in United States
Anti-Tau is a laboratory product developed by Merck Group. It is designed to detect and quantify the tau protein, which is involved in the pathogenesis of neurodegenerative diseases. The core function of Anti-Tau is to provide researchers with a tool to analyze and measure tau levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti tubulin, by Merck Group (3 mentions)
Anti map2, by Synaptic Systems (2 mentions)
Anti non phospho tau, by Merck Group (2 mentions)
Anti pthr231 tau, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti mbp, by Bio-Rad (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti pp1, by Santa Cruz Biotechnology (1 mentions)
Anti pp1 pt320, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti ptau thr231, by Merck Group (1 mentions)
Anti p gsk3β ser9, by Cell Signaling Technology (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Human igf1, by Thermo Fisher Scientific (1 mentions)
Sp2 confocal microscope, by Leica (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Anti lamin a c, by Abcam (1 mentions)
Anti yap1, by Merck Group (1 mentions)
Anti ubiquitin, by Novus Biologicals (1 mentions)
11 protocols using anti tau
1
Quantitative Western Blot Analysis of Brain Proteins
2
Murine Motoneuron Culture and IGF1 Stimulation
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Murine embryonic spinal motoneurons were cultured as described [52 (link)]. BDNF, human IGF1 (PeProTech) and mouse IGFBP5 (BP5DU020, GroPep) were used at the concentrations indicated. Cells were initially counted 4 h after plating to obtain the reference value for 100 % survival. After 7 DIV, surviving motoneuron cells were counted again. For IGF1 stimulation experiments, motoneurons were cultured for 5 days with 5 ng/ml BDNF in full medium, and then starved in the same medium without horse serum and BDNF for 12 h. Cells were then stimulated with IGF1. Neurons were fixed with 4 % PFA and stained with anti-Tau (1:1000, Sigma) and anti-IGFBP5 (1:500, Neuromics) antibodies. Cellular morphology was visualized with fluorescence-coupled secondary antibodies (Jackson ImmunoResearch) using a Leica SP2 confocal microscope. Measurements were done with the Leica AS Lite software (Leica).
3
Subcellular Fractionation and Western Blot
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Different cells were harvested and fractionated to produce cytosolic and nuclear lysates using the NE-PER kit (Thermo Fisher Scientific). For whole cell lysates, cells were lysed in RIPA buffer. Both protease and phosphatase inhibitors were included in the respective lysis buffer. ~47 μg of protein was separated on a 4%-15% gradient SDS-polyacrylamide gel (Bio-Rad) which were transferred to nitrocellulose membranes, stained with Ponceau S stain, washed, blocked with 5% non-fat milk, and incubated overnight at 4°C with the following primary antibodies: anti-α-Tubulin (Cell Signaling Technology (C.S.T.) #2144), anti-Lamin A/C (C.S.T. #4777), anti-SON (Abcam #121033 and LSBio LS-C803664), anti-YAP1 (C.S.T. #12395), anti-GFP (C.S.T. #2956), anti-Tau (Sigma-Aldrich, #A0024), anti-p-Tau (a gift from Dr. Peters Davies), anti-β-actin (C.S.T. #4970), anti-puromycin (BioLegend 381502), anti-ubiquitin (C.S.T. #58395), anti-LC3-I/II (C.S.T. #2775), anti-p62 (C.S.T. #23214), anti-ATF4 (C.S.T. #11815), anti-ATF6 (Novus 70B1413.1), and anti-XBP1s (BioLegend 658802). Membranes were treated with the appropriate secondary antibody conjugated to horseradish peroxidase the following day and then ECL Prime Western Blotting Detection Reagent (Cytiva) was applied. A Bio-Rad ChemiDoc MP Imaging System was used to visualize the signal, and signal intensities were determined with ImageJ 112 (link).
4
Ultrastructural Immunogold Localization of Tau in Larval CNS
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Filleted CNS preparations from L3 larvae were fixed in 4% PFA
(15 min), then the anterior half of the fillet was cut away and
post-fixed overnight in fresh 4% PFA. Samples were cryoprotected by incubation
in a glycerol series up to 30%. Samples were briefly plunge-frozen in liquid
ethane (−170 °C). Frozen samples were
incubated in 1.5% uranyl acetate in methanol at-90 °C
for 30 h, then at −45 °C
for11 h. This was followed by infiltration with a series of
resin:methanol mixtures at −45 °C,
culminating in pure Lowicryl HM-20 resin (EM Science) overnight. Specimens were
polymerized by UV light (24 h,
−45 °C), then the temperature increased to
0 °C over 9 h. Gold/silver sections were cut
through the proximal peripheral nerves on a Leica ultramicrotome, and
transferred to formvar-coated Ni grids (EM Science). Immunochemistry was carried
out as described28 (link) using anti-tau (Sigma, polyclonal T6402
1:100), and secondary goat-anti-rabbit 10 nm gold (EM Sciences). The
specimens were viewed on a FEI Tecnai G2 Spirit Transmission Electron
Microscope.
(15 min), then the anterior half of the fillet was cut away and
post-fixed overnight in fresh 4% PFA. Samples were cryoprotected by incubation
in a glycerol series up to 30%. Samples were briefly plunge-frozen in liquid
ethane (−170 °C). Frozen samples were
incubated in 1.5% uranyl acetate in methanol at-90 °C
for 30 h, then at −45 °C
for11 h. This was followed by infiltration with a series of
resin:methanol mixtures at −45 °C,
culminating in pure Lowicryl HM-20 resin (EM Science) overnight. Specimens were
polymerized by UV light (24 h,
−45 °C), then the temperature increased to
0 °C over 9 h. Gold/silver sections were cut
through the proximal peripheral nerves on a Leica ultramicrotome, and
transferred to formvar-coated Ni grids (EM Science). Immunochemistry was carried
out as described28 (link) using anti-tau (Sigma, polyclonal T6402
1:100), and secondary goat-anti-rabbit 10 nm gold (EM Sciences). The
specimens were viewed on a FEI Tecnai G2 Spirit Transmission Electron
Microscope.
5
Immunofluorescence Analysis of Neuronal Markers
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For immunofluorescence analysis, DIV14-21 neurons in the microfluidic device were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature (RT). The fixation buffer was rinsed out with PBS, after which neurons were permeabilized with 0.2% Triton X-100 for 30 min and subsequently incubated for 30 min with a blocking solution containing 5% bovine serum albumin (BSA). Anti-Tau (Merck Millipore), anti-MAP2 (Merck Millipore), anti-synapsin (Synaptic Systems), and anti-Shank3 (Synaptic Systems) antibodies were applied to microchannels and the microfluidic device was incubated overnight at 4 °C. After incubating with primary antibodies, neurons were washed with PBS for 30 min and subsequently incubated with Alexa 488- and/or Alexa 546-conjugated secondary antibodies (Invitrogen) for 1 h, providing different color combinations as needed.
6
Immunofluorescence Staining of Cultured Neurons
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DIV 6 or DIV 24 cultured hippocampal/cortical neurons were fixed in 4% paraformaldehyde, 4% sucrose, Tyrode's solution (136 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM Na‐HEPES, 10 mM d ‐glucose, pH 7.3) for 15 min, permeabilized for 5 min in 0.25% Triton X‐100, Tyrode's solution, and then incubated in 5% Normal Donkey Serum (NDS), Tyrode's solution for 30 min at 37°C for blocking. Primary antibodies (anti‐tdTomato, Abcam, chicken, 1:5,000; anti‐MAP2, Synaptic Systems, guinea pig, 1:1,000; anti‐tau, EMD Millipore, Mouse, 1:1,000 anti‐PSD‐95, Abcam, mouse, 1:1,000, anti‐gephyrin, Synaptic Systems, mouse, 1:1,000) diluted in Tyrode's solution with 5% NDS were incubated for 2 h at 37° C. Then, appropriate secondary antibodies (Thermo Fisher Scientific, 1:1,000) diluted in Tyrode's solution with 5% NDS were incubated for 45 min at 37°C.
7
Quantitative Protein Analysis in Flies
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Western blotting was performed as described previously [59 (link)]. Ten fly heads for each genotype were homogenized in Tris-Glycine SDS sample buffer, and the same amount of the lysate was loaded to each lane of 10%, 12.5% or 15% Tris-Glycine gels and transferred to nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk, blotted with the antibodies described below, incubated with appropriate secondary antibody and developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences). The membranes were also probed with Anti-tubulin, and used as the loading control for other blots in each experiment. Anti-tubulin (Sigma-Aldrich), anti-tau (Merck Millipore), anti-non phospho tau (Merck Millipore), anti-pSer202 tau (Thermo Fisher Scientific), anti-pThr231 tau (Thermo Fisher Scientific), anti-pSer404 tau (Abcam), anti-pSer422 tau (Abcam), anti-Nervana (Developmental Studies Hybridoma Bank), anti-VDAC1 (Abcam), anti-NDUFS3 (Abcam), anti-MnSOD (Enzo Life Sciences), anti-Atg8 (Merck Millipore) and anti-ref(2)P (Abcam) antibodies were purchased. Anti-MANF is a kind gift from Dr. T. I. Heino (University of Helsinki). Imaging was performed with ImageQuant LAS 4000 (GE Healthcare Life Sciences), and the signal intensity was quantified using Image J (NIH).
8
Antibodies Used in Neuroscience Research
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The following antibodies were used in this study: Affinity-purified neuroserpin apK53, a rabbit polyclonal antibody raised to a synthetic peptide corresponding to the 19 C-terminal amino acids of rat neuroserpin [20] , anti-FLAG (Cat No: F3165, Sigma-Aldrich Co. LLC, MO, USA), anti-GAPDH (Cat No: ab8245, Abcam, Cambridge, UK), anti-synapsin (Cat No: 611392, BD Biosciences, CA, USA), anti-PSD-95 (Cat No: MAI-045, Thermo Scientific, IL, USA), anti-NR1 (Cat No: 556308, BD Biosciences, CA, USA), anti-Tau (Cat No: MAB3420, Millipore, MA, USA), anti-GAP-43 (Cat No: G9264, Sigma-Aldrich Co. LLC, MO, USA), goat anti-rabbit IgG (H+L) Horseradish Peroxidase-conjugated antibody (Cat No: 111-035-003, Jackson ImmunoResearch Laboratories, Inc., PA, USA), and goat anti-mouse IgG (H+L) Horseradish Peroxidase-conjugated (Cat No: 115-035-003, Jackson ImmunoResearch Laboratories, Inc., PA, USA).
9
Immunofluorescence Staining of Neuronal Markers
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Cells were seeded on matrigel-coated glass coverslips and they were fixed for 20 min in ice in 4% paraformaldehyde (PFA, Sigma), solution in phosphate-buffered saline (PBS, Euroclone). Then, cells were permeabilized for 30 min in a blocking solution, containing 0.5% Triton X-100 (Sigma-Aldrich) and 10% donkey serum (Sigma-Aldrich), and incubated overnight at 4 °C with the primary antibodies in a blocking solution. Then, cells were washed with PBS and incubated for 1 h at room temperature with Hoechst and with secondary antibodies. The following antibodies were used: anti-OCT4 (1:100, Abcam), anti-NANOG (1:100, Abcam), anti-FOXA2 (1:300, Abcam), anti-NESTIN (1:300 Millipore), anti-TH (1:200, Immunological Sciences), anti-MAP2 (1:500, Immunological Sciences), anti-TOMM20 (1:300, Novus), anti-α-Synuclein (clone LB509, 1:100, Thermo), anti-GFP (1:500, Thermo), anti-α-Synuclein (phosphoS129, 1:300, Abcam), anti-TAU (1:500, Millipore), anti-LAMP1 (1:500, Abcam), anti-Synapsin-1 (1:500, Synaptic Systems), anti-SMI311 (1:500, BioLegend), anti GM130 (1:300, BD), anti-GRIM19 (1:300, Abcam). All the secondary antibodies used for the immunofluorescence staining are Alexa FluorTM.
10
Immunocytochemical Staining of Neurons
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After fixation using 4% PFA for 5 min, cells were washed twice and blocked in PBS solution containing 10% horse serum, 0.5% BSA and 0.2% Triton. Cells were stained with primary antibody for 1 h: anti‐myc antibody obtained from 9E10 hybridoma lines and used as supernatant at 1:100, anti‐SV2 (Neuromab) used at 1:200, anti‐Piccolo (Synaptic Systems) used at 1:500, anti‐tau (Millipore) used at 1:1,000 and anti‐MAP2 (Synaptic Systems) used at 1:1,000. Cells were washed and stained with secondary antibody for 1 h: anti‐mouse 405 (Jackson Dylight) antibody was used at 1:500, anti‐mouse Alexa 568 (Invitrogen) was used at 1:1,000, anti‐rabbit Alexa 555 (Invitrogen) was used at 1:1,000 and anti‐guinea pig Alexa 647 (Invitrogen) was used at 1:1,000. Images of fixed cultures were taken on a Zeiss LSM700 confocal using a 63× oil objective (NA 1.4) and a 20× water objective (NA 1.0).
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