Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Tau (phospho T205) with ab254410 at 1/20000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum without alkaline phosphatase treatment (image A, PMID: 28035925). No signal was detected when tissues were treated with alkaline phosphatase (image B).The section was incubated with ab254410 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum tissue labeling Tau (phospho T205) with ab254410 at 1/500 (1.034 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on mouse cerebrum, while nearly no staining on mouse cerebrum after alkaline phosphatase (AP) treatment. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

All lanes : Anti-Tau (phospho T205) antibody [EPR23505-13] (ab254410) at 1/1000 dilution

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

Lysates/proteins at 10 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: 21722209).

Dot blot analysis of Tau (phospho T205) labeled with ab254410 at 1/1000 dilution.

Lane 1: Tau (phospho S202+T205) peptide (aa 199-211).

Lane 2: Tau (phospho S202+T205) peptide (aa 197-209).

Lane 3: Tau peptide (aa 197-211).

Lane 4: Tau (phospho S202) peptide (aa 197-211).

Lane 5: Tau (phospho T205) peptide (aa 197-211).

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.

Tau (phospho T205) was immunoprecipitated from 0.35 mg Rat brain tissue lysate with ab254410 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254410 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Rat brain tissue lysate 10 ug

Lane 2: ab254410 IP in Rat brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254410 in Rat brain tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum tissue labeling Tau (phospho T205) with ab254410 at 1/500 (1.034 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat cerebrum, while nearly no staining on rat cerebrum after alkaline phosphatase (AP) treatment. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

All lanes : Anti-Tau (phospho T205) antibody [EPR23505-13] (ab254410) at 1/1000 dilution

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

Lysates/proteins at 10 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: 48 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 21722209).

All lanes : Anti-Tau (phospho T205) antibody [EPR23505-13] (ab254410) at 1/1000 dilution

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

Lysates/proteins at 10 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: 70 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 21722209).

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling Tau (phospho T205) with ab254410 at 1/20000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human breast without alkaline phosphatase treatment (image A, PMID: 15914550). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab254410 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins.

Tau (phospho T205) was immunoprecipitated from 0.35 mg Mouse brain tissue lysate with ab254410 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254410 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10 ug

Lane 2: ab254410 IP in Mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254410 in Mouse brain tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human Alzheimer's disease cerebrum tissue labeling Tau (phospho T205) with ab254410 at 1/20000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human Alzheimer's disease cerebrum without alkaline phosphatase treatment (image A, PMID: 20631843). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab254410 for 10 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins.