Anti-Tau (MBD region) antibody [EPR25205-233]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling Tau (MBD region) with ab308439 at 1/1000 (0.482 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Negative control : no staining in human spleen. The section was incubated with ab308439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Composite multiplex immunofluorescence staining of SYP, MAP2 and Tau (MBD region) staining in a section of formalin-fixed paraffin-embedded human Alzheimer’s brain*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with EDTA (Ph9.0) using retrieval settings of 100°C for 40 minutes. The section was then incubated at room temperature for 1 hour with ab309493 at 1µg/ml dilution (shown in green), ab318993 at 1µg/ml (shown in magenta), and ab308439 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, ab150086 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) preadsorbed 1/1000, and ab150173 Goat Anti-Chicken IgY H&L (Alexa Fluor® 488) preadsorbed 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium ^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling Tau (MBD region) with ab308439 at 1/5000 (0.1 ug/ml) followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining in human Alzheimer's brain.

The section was incubated with ab308439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling Tau (MBD region) with ab308439 at 1/1000 (0.482 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in human cerebrum. The section was incubated with ab308439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling Tau (MBD region) with ab308439 at 1/5000 (0.096 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Negative control : no staining in mouse spleen. The section was incubated with ab308439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebellum (fresh) tissue labeling Tau (MBD region) with ab308439 at 1/50 (9.64 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining on rat cerebellum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab308439 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat spleen (fresh) tissue labeling Tau (MBD region) with ab308439 at 1/50 (9.64 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Negative control : confocal image showing no staining on rat spleen (PMID : 24309898). The nuclear counterstain was DAPI (Blue). The section was incubated with ab308439 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebellum (fresh) tissue labeling Tau (MBD region) with ab308439 at 1/50 (9.64 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining on mouse cerebellum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab308439 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh) tissue labeling Tau (MBD region) with ab308439 at 1/50 (9.64 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Negative control : confocal image showing no staining on mouse spleen (PMID : 24309898). The nuclear counterstain was DAPI (Blue). The section was incubated with ab308439 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neuron cells labelling Tau (MBD region) with ab308439 at 1/50 (9.64 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/mL dilution (Green). Confocal image showing cytoplasmic staining in rat primary neuron.Confocal scanning Z step was set as 0.3 ?m followed by image processing with maximum Z projection ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 4ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/mL dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling Tau (MBD region) with ab308439 at 1/50 (9.64 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/mL dilution (Green). Confocal image showing cytoplasmic staining in mouse primary neuron.Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 4ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/mL dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling Tau (MBD region) with ab308439 at 1/5000 (0.096 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in rat cerebrum. The section was incubated with ab308439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling Tau (MBD region) with ab308439 at 1/5000 (0.096 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in mouse cerebrum. The section was incubated with ab308439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Composite multiplex immunofluorescence staining of ab279297, ab317042 and ab308439 staining NeuN, NEFH and Tau (MBD region) in Mouse Primary Neurons DIV14 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab279297 (shown in green), ab317042 (shown in magenta) and ab308439 (shown in yellow) at 1µg/ml. Cells were then incubated with ab150161 Goat F(ab')2 Anti-Rat IgG Fc (Alexa Fluor® 488) preadsorbed, ab150175 Goat Anti-Chicken IgY H&L (Alexa Fluor® 647) preadsorbed and ab150084 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized rat primary neuron cells labelling Tau (MBD region) with ab308439 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized mouse primary neuron cells labelling Tau (MBD region) with ab308439 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Tau (MBD region) was immunoprecipitated from 0.35 mg Mouse brain tissue lysate with ab308439 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab308439 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse brain tissue lysate
Lane 2 : ab308439 IP in Mouse brain tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab308439 in mouse brain tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-Tau (MBD region) antibody [EPR25205-233] (ab308439) at 1/1000 dilution

All lanes:

Mouse brain tissue lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 33 kDa,50 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : spleen (PMID : 24309898; PMID : 24386422), kidney (PMID : 24386422). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200,000 dilution. Exposure times : Lane 1 : 26 seconds; Lanes 2-4 : 81 seconds.

All lanes:

Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (ab308439) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Human cerebellum tissue lysate at 20 µg

Lane 3:

Human kidney tissue lysate at 20 µg

Lane 4:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 78 kDa

Observed band size: 33-50 kDa

false

• WB

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Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution. Exposure time : 8 seconds.

All lanes:

Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (ab308439) at 1/1000 dilution

Lane 1:

HEK-293 transfected with Tau 0N3R (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

HEK-293 transfected with Tau 1N3R (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

HEK-293 transfected with Tau 2N3R (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 4:

HEK-293 transfected with Tau 0N4R (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 5:

HEK-293 transfected with Tau 1N4R (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 6:

HEK-293 transfected with Tau 2N4R (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 78 kDa

Observed band size: 10-70 kDa

false

Exposure time: 8s

• AP

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Affinity Purification - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Biotinylated Human Tau 2N4R (Protein : P9954) [1μg/ml] is loaded to SA biosensor on Octet RED96e machine, then associate with Anti-Tau (MBD region) antibody [EPR25205-233] (ab308440) in serial concentration points [53.35, 26.68, 13.34, 6.67, 3.34 nM/ml] by 2-fold dilution, then dissociate in blank testing buffer [10mM HEPES (0.05% Tween-20, 3mM EDTA, 100mM NaCl)]. Calculated signals had already subtracted blank control, curve fitting using 1 : 1 binding model. K_D (M) value of Anti-Tau (MBD region) antibody [EPR25205-233] (ab308440) is 4.78E-10.

• WB

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Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : testis (PMID : 24386422). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200, 000 dilution. Exposure time : 180 seconds.

All lanes:

Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (ab308439) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat testis tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 50-70 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (AB308439)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : spleen (PMID : 24309898; PMID : 24386422).

In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10, 000 dilution.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (ab308439) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 50 kDa

true

Exposure time: 180s