Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling Tau (phospho T181) with ab254409 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse cerebrum tissue labeling Tau (phospho T181) with ab254409 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on mouse cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

All lanes : Anti-Tau (phospho T181) antibody [EPR23506-107] (ab254409) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse brain tissue lysate (phosphatase treated membrane)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 78 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

The molecular weight observed is consistent with what has been described in the literature (PMID: 21437732).

This blot was developed using a higher sensitivity ECL substrate.

Tau (phospho T181) was immunoprecipitated from 0.35 mg of mouse brain tissue lysate with ab254409 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab254409 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10μg (Input). 
Lane 2: ab254409 IP in mouse brain tissue lysate. 
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254409 in mouse brain tissue lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5.5 seconds.

All lanes : Anti-Tau (phospho T181) antibody [EPR23506-107] (ab254409) at 1/1000 dilution

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat brain tissue lysate (phosphatase treated membrane)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 78 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

The molecular weight observed is consistent with what has been described in the literature (PMID: 21437732).

This blot was developed using a higher sensitivity ECL substrate.

Tau (phospho T181) was immunoprecipitated from 0.35 mg of rat brain tissue lysate with ab254409 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab254409 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: Rat brain tissue lysate 10μg (Input).
Lane 2: ab254409 IP in rat brain tissue lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254409 in rat brain tissue lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat cerebrum tissue labeling Tau (phospho T181) with ab254409 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat cerebrum without alkaline phosphatase treatment (image A). Nearly no signal was detected when tissues were treated with alkaline phosphatase (image B). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling Tau (phospho T181) with ab254409 at 1/5000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum without alkaline phosphatase treatment (image A, PMID: 30279741). No signal was detected when tissues were treated with alkaline phosphatase (image B). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Antigen: Mouse Tau non-phospho peptide，Mouse Tau (phospho T181) peptide.

Antigen concentration: 1000 ng/mL.

ab254409 used at 0 - 2000 ng/mL.

Secondary antibody: Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution.

Dot blot analysis of Tau (phospho T181) labeled with ab254409 at 1/1000 dilution.

Lane 1: Tau (phospho T181) peptide (aa 177-190).
Lane 2: Tau non-phospho peptide (aa 174-190).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.