Anti-Tau (phospho S396) 抗体 [E178]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [E178] (AB32057)

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.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tau (phospho S396) antibody [E178] (AB32057)

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

This image is courtesy of Carl Hobbs, King's College London, United Kingdom

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Tau (phospho S396) antibody [E178] (AB32057)

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.

• IP

Unknown

Immunoprecipitation - Anti-Tau (phospho S396) antibody [E178] (AB32057)

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.

All lanes:

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

Predicted band size: 78 kDa

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• ELISA

Lab

ELISA - Anti-Tau (phospho S396) antibody [E178] (AB32057)

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.

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-Tau (phospho S396) antibody [E178] (ab32057) at 1/1000 dilution

Lane 1:

Human brain lysates 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

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Exposure time: 60s

• AP

Lab

Affinity Purification - Anti-Tau (phospho S396) antibody [E178] (AB32057)

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

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-Tau (phospho S396) antibody [E178] (ab32057) at 1/1000 dilution

Lane 1:

Mouse brain lysates 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 [E178] (AB32057)

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.

• WB

CiteAb

Western blot - Anti-Tau (phospho S396) antibody [E178] (AB32057)

Tau (phospho S396) western blot using anti-Tau (phospho S396) antibody [E178] ab32057. Publication image and figure legend from Zhang, R., Liu, Y., et al., 2020, CNS Neurosci Ther, PubMed 31364823.

ab32057 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32057 please see the product overview.

Analysis of glymphatic transport and brain macromolecule levels of WT and AQP4 KO mice following sleep disruption (SD). A, Representative images showing TR-d3 fluorescence in six coronal brain sections from bregma +1.0 mm to -1.5 mm at 30 min after the start of injection into the cisterna magna. B, Representative images of coronal slices at bregma +0.5 mm from four mice per group showing TR-d3 distribution within the brain parenchyma at the time point of 45 min. C, Quantification of the percentage area of TR-d3 fluorescence in whole brain section of four groups. D, Diagram showing the subregional analysis of brain sections at the levels of bregma +0.5 mm. E, Quantification of the TR-d3 fluorescence intensity (AU, arbitrary units) of the dorsal, ventral, and lateral brain regions at 30-min time point, respectively. F, Quantification of the percentage area of TR-d3 fluorescence in the coronal brain section at bregma anterior to 0.5 mm, at 30 and 45 min after the start of injection. G, H, Representative Western blot bands and densitometry analysis of the expression levels of hippocampal Aβ1-40, APP, total Tau, and hyperphosphorylated Tau at Ser396/404 that are measured with PHF-1 antibodies. I, ELISA analysis of soluble Aβ1-40 levels from the hippocampal samples. J, K, Representative Western blot bands and densitometry analysis of the expression levels of Aβ generation, clearance, and transport-related markers from the hippocampal samples. Data in (C) were analyzed by repeated-measures ANOVA and in the other figures were analyzed by the two-way ANOVA, with Tukey's post hoc test. Data represent mean ± SEM from four mice per group. *P < .05, **P < .01, SD vs Con; #P < .05, ##P < .01, AQP4 KO vs WT. AQP4, aquaporin 4; KO, knockout; WT, wild-type

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