Recombinant 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.

Blocking/Diluting buffer and concentration 5% NFDM/TBST

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

Diluting/Diluting buffer and concentration 5% NFDM/TBST

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

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

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.

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.

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.

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 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 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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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.