The following anti-tau antibodies were used in this study: RD4 (1:1,000, 05–804, Millipore), Tau5 (1:4,000, MA5-12808, ThermoFisher), 2B11 (1:500, 10237, IBL), AT8 (1:1,000, MN1020, ThermoFisher), AT100 (1:1,000, MN1060, Invitrogen), Ser262 (1:2,000, ab64193, Abcam), and pS396 (1:2,000, ab109390, Abcam). The mouse monoclonal antibody (mAb) RD4 (directed to amino acids 275–291 of 4-repeat [4R] tau) preferentially recognizes 4R isoform of tau [37 ]. The mAbs 2B11 (directed to residues 306–312) and Tau5 (recognizing amino acids 218–225) react with both 3R and 4R isoforms of tau. The rabbit polyclonal serum Ser262 reacts with both non-phosphorylated and phosphorylated serine at residue 262. Phosphorylation-dependent antibodies AT8, AT100 and pS396 recognizes pSer202/pSer205, pThr212/pSer214/pThr217, and pSer396, respectively [38 ,39 ].
Mn1060
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MN1060 is a laboratory centrifuge designed for general-purpose applications. It is capable of reaching a maximum speed of 6,000 rpm and can accommodate a variety of sample tube sizes. The centrifuge features a digital display and user-friendly controls for setting speed, time, and temperature.
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Lab products found in correlation
Mn1020, by Thermo Fisher Scientific (3 mentions)
Ab79415, by Abcam (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bio-Rad (2 mentions)
Sudan black b, by Merck Group (2 mentions)
Ma532569, by Thermo Fisher Scientific (2 mentions)
Ma529010, by Thermo Fisher Scientific (2 mentions)
Alexa fluor fluorescently conjugated secondary goat anti rabbit and anti mouse antibodies, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ab64193, by Abcam (1 mentions)
Ab109390, by Abcam (1 mentions)
Af1729, by R&D Systems (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab108319, by Abcam (1 mentions)
Af1828, by R&D Systems (1 mentions)
Trueblack solution, by Biotium (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade with dapi, by Thermo Fisher Scientific (1 mentions)
8 protocols using mn1060
1
Tau Isoform-Specific Antibodies Analysis
2
Hippocampal Protein Profiling Methodology
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Hippocampal tissue was extracted after decapitation and stored frozen. Brain tissue processing followed established protocols.59 Total protein concentration was quantified by standard BCA assay (Pierce). Equal amounts of protein (25 μg/μL) were separated by SDS‐PAGE on 10% gels and were then transferred to nitrocellulose membranes (0.45 μm; Thermo Fisher Scientific). The membranes were blocked for 2 hours with 5% non‐fat dry milk or BSA at room temperature, then incubated at 4°C overnight with the following antibodies: brain‐derived neurotrophic factor (BDNF; ab108319; Abcam, 1:2000), TREM2 (AF1729, R&D Systems, 1:5000), interleukin 1α (IL‐1α; OTI2F8, Novus Biologicals, 1:2000), human leukocyte antigen‐DR isotype (HLA‐DR; sc‐53319, Santa Cruz Biotechnology, 1:5000), anti‐phospho‐Tau (AT8; MN1020, Invitrogen, 1:1000), and paired helical filaments (PHF; MN1060, Invitrogen, 1:1000). After washing, the membranes were incubated for 1.5 hours at room temperature, with either horseradish peroxidase–labeled anti‐rabbit immunoglobulin G (IgG; 31460) or anti‐mouse IgG (31430, Thermo Fisher Scientific, 1:10000). The protein bands were visualized using chemiluminescent substrate (34577, Thermo Fisher Scientific) and quantified with ImageJ. Each sample was normalized to Ponceau staining of total proteins.
3
Immuno-profiling of Tauopathy in Tissue
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50 μm‐thick free‐floating sections were incubated in an antigen retrieval solution (Wako, S1700) at 60°C for 30 minutes. After that, sections were incubated in blocking solution: 5% donkey serum, 5% goat serum, 5% bovine serum albumin in PBS 0.3% triton for 2 hours at room temperature under agitation. Sections were then incubated for 48 hours with the following antibodies: IBA‐1 (234‐006, Synaptic Systems, 1:1000), AT8 (MN1020, Invitrogen, 1:500), AT100 (MN1060, Invitrogen, 1:1000), pTau 422 (ab79415, Abcam, 1:1000), NeuN (266004, Syn. Systems, 1:1000), TREM2 (AF1828, R&D systems, 1:500), MAP2 (188004, Syn, Systems, 1:1000), Tau 3R (2A1‐1F4, Wako, 1:400), Tau 4R (ab218314, Abcam, 1:500), CP3, MC1, and PHF1 (provided by Dr. Peter Davies, 1:400). TOC1 antibody for tau oligomers was provided by Dr. Nicholas Kanaan and used at 1:1000. Tissue was washed thoroughly with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen, 1:500) for 2 hours, at room temperature. True Black solution (Biotium) was used for 30 minutes to eliminate lipofuscin autofluorescence. Slides were then mounted with Prolong Diamond Antifade with Dapi (Invitrogen).
4
Immunostaining of Phospho-Tau and Antioxidant Proteins
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Deparaffinization and antigen retrieval of the brain tissue sections were performed as previously described.30 (link) After washing with phosphate-buffered saline (PBS), sections were blocked in 0.1% Triton X-100 (Sigma-Aldrich), 2% normal goat serum (Thermo Fisher Scientific, MA, USA) and 1% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies diluted in blocking buffer overnight at +4°C. After 3 × 10 min washes in 0.1% Triton X-100 in PBS, they were incubated with secondary antibodies and DAPI (2-(4-Amidinophenyl)-1H-indole-6-carboxamidine) for 1 h at room temperature followed by 3 × 10 min washes in Triton X-100 in PBS. The following dilutions and antibodies were used: 1:100 mouse anti-phospho-tau (Thr212, Ser214) (MN1060; Thermo Fisher Scientific), 1:100 rabbit anti-TRX-1 (MA532569; Thermo Fisher Scientific), 1:100 rabbit anti-AGT (MA529010; Thermo Fisher Scientific), 1:500 Alexa Fluor fluorescently conjugated secondary goat anti-rabbit and anti-mouse antibodies (Thermo Fisher Scientific) and 1:100 DAPI (1351303; Bio-Rad, USA). To quench the autofluorescence of lipofuscin, sections were washed for 5 min in PBS and incubated with 0.1% w/w Sudan Black B (199664; Sigma-Aldrich) in 70% ethanol. Prior to mounting, sections were washed with Triton X-100 in PBS 2 × 10 min and 10 min with PBS.
5
Immunostaining of Phospho-Tau and Antioxidant Proteins
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Deparaffinization and antigen retrieval of the brain tissue sections were performed as previously described.30 (link) After washing with phosphate-buffered saline (PBS), sections were blocked in 0.1% Triton X-100 (Sigma-Aldrich), 2% normal goat serum (Thermo Fisher Scientific, MA, USA) and 1% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies diluted in blocking buffer overnight at +4°C. After 3 × 10 min washes in 0.1% Triton X-100 in PBS, they were incubated with secondary antibodies and DAPI (2-(4-Amidinophenyl)-1H-indole-6-carboxamidine) for 1 h at room temperature followed by 3 × 10 min washes in Triton X-100 in PBS. The following dilutions and antibodies were used: 1:100 mouse anti-phospho-tau (Thr212, Ser214) (MN1060; Thermo Fisher Scientific), 1:100 rabbit anti-TRX-1 (MA532569; Thermo Fisher Scientific), 1:100 rabbit anti-AGT (MA529010; Thermo Fisher Scientific), 1:500 Alexa Fluor fluorescently conjugated secondary goat anti-rabbit and anti-mouse antibodies (Thermo Fisher Scientific) and 1:100 DAPI (1351303; Bio-Rad, USA). To quench the autofluorescence of lipofuscin, sections were washed for 5 min in PBS and incubated with 0.1% w/w Sudan Black B (199664; Sigma-Aldrich) in 70% ethanol. Prior to mounting, sections were washed with Triton X-100 in PBS 2 × 10 min and 10 min with PBS.
6
Immunodetection of Tau Protein Phosphorylation
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To detect tau by immunostaining, we used biotin-conjugated anti-pS202/pT205 tau (AT8; 1:500; mouse monoclonal, catalog #MN1020B, Thermo Fisher Scientific) and anti-pT212/pS214/pT217 tau (AT100; 1:500; mouse monoclonal, MN1060, Thermo Fisher Scientific). The following additional antibodies were used for immunostaining: anti-Aβ (D12B2; 1:1000; rabbit monoclonal; catalog #9888, Cell Signaling Technology) and anti-NeuN (1:500; mouse monoclonal; catalog #MAB377, Millipore). For immunoblotting, we used the following antibodies: anti-mouse tau (T49; 1:50,000; mouse monoclonal; catalog #MABN827, Millipore), anti-APP (22C11; 1:2500; mouse monoclonal; catalog #MAB348, Millipore), anti-GAPDH (1:10,000; mouse monoclonal; catalog #MAB374, Millipore), AT8 (1:500), AT100 (1:500), anti-pS422 tau (1:1000; rabbit monoclonal; catalog #ab79415, abcam), anti-tau (BR133; 1:4000; rabbit polyclonal, in-house), and anti-tau (BR134; 1:4000; rabbit polyclonal, in-house). For immunoelectron microscopy, we used the following antibodies: anti-mouse tau (MT1; 1:50; rabbit polyclonal, in-house), BR134 (1:50), AT8 (1:50), and AT100 (1:50).
7
Analyzing Protein Expression in Hippocampi
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Protein samples were isolated from hippocampi or cells using RIPA lysis buffer (150 mM NaCl, 1% Triton™ X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris pH8.0) supplemented with PhosSTOP™ and protease inhibitors (Roche). Protein levels were determined by resolving 20 μg of protein on SDS-PAGE gels, transferred onto nitrocellulose or PVDF membranes and incubated with primary antibodies for eIF2α-P (1:1000; Cell Signaling 3597s), eIF2α (1:1000; Cell Signaling 2103s), ATF4 (CREB-2, 1:1000; Santa Cruz sc200), ATF6 (1:1000; Genetek 70B1413), GSK3β (1:2000, Cell Signaling, 9832), pSer9-GSK3β (1:1000, Cell Signaling, 9322), total tau (tau-5, 1:2000; Invitrogen ANB0042), p-tau (AT100, 1:2000; Thermo Fisher Scientific MN1060). Horseradish peroxidase-conjugated secondary antibodies (1:5000; Dako) were applied and protein visualized using enhanced chemiluminescence (GE Healthcare) and quantitated using ImageJ. Antibodies against GAPDH (1:5000; Santa Cruz sc32233), β-actin (1:5000; Abcam ab8227) and β-tubulin (1:5000; Millipore MAB1637) were used to determine loading. To detect PrPSc (prion protein) homogenized samples were digested with 50 μg/ml of proteinase K (PK) at 37°C for 1 h prior to electrophoresis. Membranes were then probed with ICSM-35 (1:10 000; D-GEN 0130-03501) and goat anti-mouse (1:10 000; Dako).
8
Immunohistochemical Analysis of Brain Tissue
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After deparaffinized and gradual hydration through graded alcohols, paraffin sections of brain tissue were immersed in EDTA antigen retrieval buffer (pH 6.0) and maintained at a sub-boiling temperature for 9 min, standing for 7 min and then followed by another sub-boiling temperature for 7 min. Sections were washed three times for 5 min each in PBS (pH 7.4). 3% bovine serum albumin was added to block non-specific binding for 30 min. Sections were incubated with the P-tau (Thr212/Ser214) primary antibody (1:200, MN1060; Thermo Fisher Scientific, Waltham, MA, USA), Aβ primary antibody (1:200, GB11197; Wuhan Servicebio Technology Co., Ltd.), or P-tau (Thr205/Ser202) primary antibody (1:200, GB113883; Wuhan Servicebio Technology Co., Ltd.) overnight at 4°C, washed three times for 5 min each in PBS, and incubated with secondary antibodies (HRP labeled) goat anti-rabbit (1:200, GB23303; Wuhan Servicebio Technology Co., Ltd.) or goat anti-mouse (1: 200, GB23301; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 50 min. After three 5-min washed in PBS, DAB chromogenic agent (G1211; Wuhan Servicebio Technology Co., Ltd.) was added. After nucleus counterstaining and dehydration through graded alcohols, sections were sealed and examined with a microscope.
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