Immunohistochemical analysis of paraffin-embedded Human endometrium carcinoma tissue labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on tumor cells of the endometrium carcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: TDP43 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab190963 observed at 48 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab190963 was shown to specifically react with TDP43 in wild type cells as signal was lost in TDP43 knockout cells. Wild-type and TDP43 knockout samples were subjected to SDS-PAGE. ab190963 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab190963 at 1/2000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling TDP43 with ab190963 at 1/1000 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] -Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

Anti-TDP43 antibody [EPR18554] (ab190963) at 1/2000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-TDP43 antibody [EPR18554] (ab190963) at 1/10000 dilution

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID:22659571, PMID:20736350).

All lanes : Anti-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID:22659571, PMID:20736350).

Anti-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution + Zebrafish head lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID:22659571, PMID:20736350).

All lanes : Anti-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 1µM staurosporine for 4 hours whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 50 µM Z-VAD-FMK for 4 hours whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 1µM staurosporine and 50 µM Z-VAD-FMK for 4 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID:22659571, PMID:20736350), and can be induced by staurosporine.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on normal Human pancreas is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on K562 cell line.

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab190963 at 1/2000 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

TDP43 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab190963 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab190963 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate, 10µg (Input).

Lane 2: ab190963 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab190963 in HeLa whole cell lysate.

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

Exposure time: 1 second.

The band of ~35 kDa is the cleavage product from caspase activity (PMID:22659571, PMID:20736350).