AHB0042

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Tau-5 was performed by loading 20 µg of Mouse brain tissue lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. Tau-5 was detected at ~50 kDa using Tau-5 Mouse Monoclonal Antibody (Product # AHB0042) at 0.5-1 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Mouse HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Tau Monoclonal Antibody (TAU-5) (Product # AHB0042) and 40-80 kDa bands corresponding to Tau were observed in Mouse and Rat Brain but not in Mouse Kidney or Mouse and Rat Lung. Tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2), Mouse Kidney (Lane 3), Mouse Lung (Lane 4) or Rat Lung (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:500) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Additional band at 25 kDa corresponds to tissue IgG and has been marked with an asterisk.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Tau in human iPSC-derived forebrain organoids derived at Day 40. The organoids were fixed with 4% PFA for 1 hour at room temperature, followed by incubation with 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT and cryosectioned at 5 µm, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were stained with a Mouse Tau monoclonal antibody (green; Product # AHB0042) at a dilution of 1:500 in blocking buffer overnight at 4°C, and then incubated with Donkey anti-Mouse Alexa Fluor 488 (Product # R37114) at a dilution of 1:1000 as well as DAPI (blue; 1:25000) in blocking solution at room temperature for 1 hour. Images were taken at 20X magnification. Data courtesy of Dr. Zhexing Wen at Emory University.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Tau showing staining in the cytoplasm of paraffin-embedded human astroglioma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Tau (Product # AHB0042) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Tau showing staining in the cytoplasm of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Tau (Product # AHB0042) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Tau showing staining in the cytoplasm and weak nuclear staining of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Tau (Product # AHB0042) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of p-Tau (S396) and Tau in human iPSC-derived forebrain organoids derived at Day 40. The organoids were fixed with 4% PFA for 1 hour at room temperature, followed by incubation with 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT and cryosectioned at 5 µm, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were stained with a Mouse Tau (TAU-5) monoclonal antibody (green; Product # AHB0042) at a dilution of 1:500 and a Rabbit p-Tau (Ser396) polyclonal antibody (red; Product # 44-752G) at a dilution of 1:500 in blocking buffer overnight at 4°C, and then incubated with Donkey anti-Mouse Alexa Fluor 488 (Product # R37114), Donkey anti-Rabbit Alexa Fluor 568 (Product # A10042) at a dilution of 1:1000 as well as DAPI (blue; 1:25000) in blocking solution at room temperature for 1 hour. Images were taken at 20X magnification. Scale bar: 50 µm. Data courtesy of Dr. Zhexing Wen at Emory University.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Tau phosphorylation precludes the BIN1-Tau interaction in vitro . a . Tau-F5 fragment phosphorylation analysis after in vitro phosphorylation by CDK2/CycA3 kinase. Representative immunoblots using various antibodies against phosphorylation epitopes in Tau. The total amount of Tau-F5 was revealed by the phosphorylation-independent antibody Tau5. The significant shift in molecular weight observed in CDK2/CycA3-treated samples indicates Tau hyperphosphorylation. In contrast to other antibodies, the Tau-1 antibody binds to various non-phosphorylated Tau sites; the signal thus decreases when Tau is hyperphosphorylated [ 31 ]. b . 2D [ 1 H, 15 N] HSQC spectra of 125 muM 15 N CDK-phosphorylated Tau-F5[165-245] free in solution (gray) and with a 1 molar amount of GST-BIN1/SH3 (red, superimposed): Overlaid details of 2D [ 1 H, 15 N] HSQC spectra presented in Additional file 6 . c . 2D [ 1 H, 15 N] HSQC spectra of 60 muM 15 N CDK-phosphorylated 2N4R Tau free in solution (gray) and with a 2 molar amount of GST-BIN1/SH3 (blue, superimposed): Overlaid details of full spectra presented in Additional file 7

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Tau phosphorylation in vitro . One microgram recombinant human tau (left, A) or mouse tau (right, A) was incubated with glycogensynthase-kinase-3beta (GSK-3ss) and ATP in kinase buffer and analyzed by Western blot using antibodies against total tau (tau-5) phospho-tau-S199, phospho-tau-T231 and phospho-tau-S396. Lanes B and C served as a control omitting either the enzyme GSK-3beta (Lane B) or tau protein (Lane C). Size markers are given as kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Western blot for p-tau S199. Brain slices of adult WT mice (A) or transgenic (TG) mice (B) were incubated for 14 days at 37degC without (Co) or with Okadaic acid (OA), wortmannin (WM), or combination of OA+WM. Extracted brain slices were subsequently analyzed by Western blot using antibodies against total tau (tau-5) and phospho-tau-S199. Size markers are given as kDa on the right side. Actin served as a control.