Recombinant Anti-Tau (phospho S396) antibody [E178] (ab32057)

Blocking/Diluting buffer and concentration 5% NFDM/TBST

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

Immunohistochemistry analysis of paraffin-embedded human cerebrum tissue sections labeling Tau (phospho S396) with ab32057 at 1/4000 dilution (0.026 μg/mL). Goat Anti-Rabbit IgG H&L (HRP polymer) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).

Positive staining on human cerebrum without alkaline phosphatase treatment (image A). No staining on human cerebrum with alkaline phosphatase treatment (image B).
The section was incubated with ab32057 overnight at +4°C.

Immunohistochemistry analysis of frozen mouse cerebrum tissue sections labeling Tau (phospho S396) with ab32057 at 1/100 (1μg/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.

ab32057 (purified) at 1/20 dilution (0.5ug) immunoprecipitating Tau in Human fetal brain lysates.
Lane 1: Human fetal brain lysates 10ug
Lane 2 (+): ab32057 & Human fetal brain lysates
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32057 in Human fetal brain lysates
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Dot blot analysis of Tau (phospho S396) phospho peptide (Lane 1) and Tau non-phospho peptide (Lane 2) labelling Tau (phospho S396) with ab32057 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Indirect ELISA antigen dose-response curve using ab32057 at 1000-0 ng/mL. Antigen Human Tau (phospho S396) peptide, Human Tau non-phospho peptide at concentration of 1000 ng/mL. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG H+L at 1/2500 dilution was used as the secondary antibody.

Blocking/Diluting buffer and concentration 5% NFDM/TBST

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

Immunohistochemistry analysis of paraffin-embedded mouse colon tissue sections labeling Tau (phospho S396) with ab32057 at 1/4000 dilution (0.026 μg/mL). Goat Anti-Rabbit IgG H&L (HRP polymer) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).

Positive staining on ganglions of mouse colon without alkaline phosphatase treatment (image A). No staining on ganglions of mouse colon with alkaline phosphatase treatment (image B).
The section was incubated with ab32057 overnight at +4°C.

Immunohistochemistry analysis of frozen rat cerebrum tissue sections labeling Tau (phospho S396) with ab32057 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 rat cerebrum, the signal decreased after phosphatase treatment at 37℃ for 2h.

Immunohistochemistry analysis of paraffin-embedded rat colon tissue sections labeling Tau (phospho S396) with ab32057 at 1/4000 dilution (0.026 μg/mL). Goat Anti-Rabbit IgG H&L (HRP polymer) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).

Positive staining on ganglions of rat colon without alkaline phosphatase treatment (image A). No staining on ganglions of rat colon with alkaline phosphatase treatment (image B).
The section was incubated with ab32057 overnight at +4°C.

IHC image of Tau 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 ab32057, 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.

Biotinylated Human Tau (pS396) [0.05 µg/ml] was loaded to SA biosensor on Fortebio RED96e Machine, then associate with recombinant Anti-Tau (phospho S396) antibody [E178] in serial concentration points [3.33, 1.67, 0.83, 0.42, 0.21 nM/mL] by 2-fold dilution, next to dissociate in blank testing buffer [0.1% BSA in PBST (0.05%Tween-20)]. Calculated signals had already subtracted blank control, curve fitting using 1:1 binding model. Non-phospho Tau protein’s association and dissociation were also showed in graph. KD(M) value of Anti-Tau (phospho S396) antibody [E178] is 2.08E-11

Immunohistochemistical detection of Tau antibody [E178] (ab32057) on formaldehyde fixed paraffin-embedded rat tongue sectionsAntigen retrieval step: Heat mediated. Buffer Used: Citric acid pH6. Permeabilization: None. Primary antibody incubated at 1/1000 for 2 hours @ 21°C in TBS/BSA/azide. Secondary antibody: anti Rabbit IgG Conjugated to Biotin (1/200). A strong pattern of immunostaining which appears to be mostly localised to nerve fibres and their cell bodies (Islet of Langerhans cells are also positive). In submitted image of central area of tongue coloured arrowheads indicate features: red for nerves cut in cross-section (T/S), each brown dot representing a single axon green for what appears to me to be small nerve fibres wrapping around a partial muscle fibre black for a Ganglion containing seven positive nerve cell bodies. Surrounding these are collagen fibres (C), adipocytes (A) and skeletal/striated muscle fibres in L/S ( M-

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Immunohistochemistical detection of Tau antibody [E178] (ab32057) on formaldehyde-fixed paraffin-embedded human salivary gland sections. Antigen retrieval step: Heat mediated.  Buffer Used: Citric acid pH6. Permeabilization: No.  Blocking step: 1% BSA for 10 mins @ 21°C. ab32057 incubated at 1/1000 for 2 hours @  21°C in TBS/BSA/azide. Secondary antibody:  anti rabbit IgG conjugated to Biotin (1/200). NB: An interesting pattern of positivity that seems to be supported by the Human Protein Atlas. Coloured arrowheads in the submitted image indicate features: red for positive serous glands, blue for positive intra-lobular collecting ducts, black for negative mucous glands (there is a serous demilune around this acinus), yellow for intralobular collecting ducts, green for nerve tracks in the interlobular areas, blue for positive interlobular collecting ducts. There appears to be a population of positive nuclei but this may b