Anti-Tau (phospho S396) 抗体 [EPR2731]

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

IHC image of Tau (phospho S396) staining in a section of frozen normal human Alzheimer brain performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab109390, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue* labelling Tau (phospho S396) with ab109390 at 0.1ug/ml followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human cerebral cortex.

The section was incubated with ab109390 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Immunohistochemistry analysis of paraffin-embedded human colon tissue sections labelling Tau (phospho S396) with ab109390 at 1/4000 dilution (0.026 μg/mL). The section was incubated with ab109390 for 30 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. Positive staining on ganglions of human colon without alkaline phosphatase treatment (image A); No signal was detected when tissues were treated with alkaline phosphatase (image B).The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Immunohistochemical staining of paraffin embedded human glioblastoma with purified ab109390 at a dilution of 1/4000. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained wirh hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• IP

Lab

Immunoprecipitation - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

ab109390 at 1/20 immunoprecipitating Tau (phospho S396) in Human brain lysate.

Lane 1 (input) : Human brain lysate (10μg)

Lane 2 (+) : ab109390 + Human brain lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109390 in Human brain lysate.

For western blotting, ab109390 at 1/1000 dilution followed by VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/1000 dilution

Predicted band size: 78 kDa

Observed band size: 50-70 kDa

false

Exposure time: 3min

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Immunohistochemistry analysis of frozen rat cerebrum tissue sections labeling Tau (phospho S396) with ab109390 at 1/100 (1 μg/mL). ab150077 Alexa Fluor^®488 Goat anti-Rabbit at 1/1000 (2 μg/mL) was used as the secondary antibody. Sections were fixed with 4% PFA and permeabilised with 0.2% Triton X-100. DAPI (blue) was used as nuclear counterstain. Antigen retrieval was heat mediated using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Cytoplasmic staining on rat cerebrum, the signal decreased after phosphatase treatment at 37℃ for 2h.

• IHC-P

AbReview39770****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

IHC image of Tau (phospho S396) staining in human Alzheimer hippocampus formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with citrate buffer. The section was then incubated with unpurified ab109390 at 1/1000 dilution for 2 hoursat 21°C. A biotin conjugated goat-anti-rabbit antibody was used as a secondary at 1/250. The section shows clear neurofibrillary tangles in a subset of neurons.

Image courtesy of Carl Hobbs, Kings College London, U.K.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Immunohistochemistry analysis of frozen mouse cerebrum tissue sections labeling Tau (phospho S396) with ab109390 at 1/100 (1 μg/mL). ab150077 AlexaFluor^®488 Goat anti-Rabbit at 1/1000 (2 μg/mL) was used as the secondary antibody. Sections were fixed with 4% PFA and permeabilised with 0.2% Triton X-100. DAPI (blue) was used as nuclear counterstain. Antigen retrieval was heat mediated using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Cytoplasmic staining on mouse cerebrum, the signal decreased after phosphatase treatment at 37℃ for 2h.

• WB

Lab

Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/20000 dilution

Lane 1:

Untreated SH-SY5Y at 10 µg

Lane 2:

SH-SY5Y treated with alkaline phosphatase at 10 µg

Secondary

All lanes:

HRP goat ant-rabbit (H+L) at 1/1000 dilution

Predicted band size: 78 kDa

Observed band size: 50-70 kDa

false

• WB

Lab

Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Blocking/Diluting buffer and concentration 5% NFDM/TBST

Tau assembles into oligomers as described in PMID : 28382304, 32692785 and 30120733.

All lanes:

Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/1000 dilution

Lane 1:

Human brain lysate at 15 µg

Lane 2:

Human brain lysates and the membrane was incubated with alkaline phosphatase at 15 µg

Lane 3:

Human brain lysates and the membrane was incubated with lambda phosphatase at 15 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: 50-79 kDa

false

Exposure time: 100s

• WB

Lab

Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Diluting/Diluting buffer and concentration 5% NFDM/TBST

Tau assembles into oligomers as described in PMID : 28382304, 32692785 and 30120733..

All lanes:

Western blot - Anti-Tau (phospho S396) antibody [EPR2731] (ab109390) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 15 µg

Lane 2:

Mouse brain lysates and the membrane was incubated with alkaline phosphatase at 15 µg

Lane 3:

Mouse brain lysates and the membrane was incubated with lambda phosphatase at 15 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: 50-79 kDa

false

Exposure time: 10s

• Dot

Lab

Dot Blot - Anti-Tau (phospho S396) antibody [EPR2731] (AB109390)

Dot blot analysis of Tau (phospho S396) phospho peptide (Lane 1) and Tau non-phospho peptide (Lane 2) labeling Tau (phospho S396) with ab109390 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG) (H+L) at 1/100 000 was used as the secondary antibody.

Blocking and diluting buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.