All lanes : Anti-Tau (phospho T231) antibody [EPR2488] (ab151559) at 1/1000 dilution (unpurified)

Lane 1 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate, untreated
Lane 2 : SH-SY5Y cell
lysate, treated with sorbitol

Lysates/proteins at 10 µg per lane.

Predicted band size: 46 kDa

IHC image of Tau (phospho T231) staining in a section of frozen normal human Azheimer 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 ab151559, 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.

Immunohistochemistry (Frozen sections) analysis of mouse cerebrum tissue sections labeling Tau with Purified ab151559 at 1/50 (1.5 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat cerebrum tissue sections labeling Tau with Purified ab151559 at 1:500 dilution (0.24 µg/ml). Heat mediated antigen retrieval was performed using citrate (pH 6.0)

ab151559 (purified) at 1:20 dilution (0.6µg) immunoprecipitating Tau in Rat cerebral cortex lysate.
Lane 1 (input): Rat cerebral cortex lysate 10µg
Lane 2 (+): ab151559 & Rat cerebral cortex lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab151559 inRat cerebral cortex lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse cerebrum tissue sections labeling Tau or Primary ab team with Purified ab151559 at 1:500 dilution (0.24 µg/ml). Heat mediated antigen retrieval was performed using citrate (pH 6.0)

All lanes : Anti-Tau (phospho T231) antibody [EPR2488] (ab151559) at 1/5000 dilution (Purified)

Lane 1 : Rat cerebral cortex lysates with 5% NFDM/TBST
Lane 2 : Rat cerebral cortex lysates. Then the membrane was incubated with phosphatase. with 5% NFDM/TBST

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 46 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cerebrum tissue sections labeling Tau with Purified ab151559 at 1:500 dilution (0.24 µg/ml). Heat mediated antigen retrieval was performed using citrate (pH 6.0)

IHC image of Tau (phospho T231) 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 ab151559 at 1/2000 dilution for 2 hours at 21ºC. 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.

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IHC image of Tau (phospho T231) staining in human Alzheimer hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with unpurified ab151559, 1/200 dilution, for 15 minutes 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.

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.

All lanes : Anti-cardiac Troponin I antibody (ab1000) at 1/1000 dilution (purified)

Lane 1 : C57 (cerebral cortex) whole cell lysate
Lane 2 : C57 (cerebral cortex) whole cell lysate incubated with phosphatase

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 46 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Tau (phospho T231) antibody [EPR2488] (ab151559) at 1/1000 dilution (purified)

Lane 1 : Human hippocampus tissue lysate
Lane 2 : Human hippocampus tissue lysate incubated with phosphatase

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 46 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking and dilution buffer: 5% NFDM/TBST.