Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Tissue Factor with ab228968 at 1/500 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human renal glomerulus (PMID: 7684196). The section was incubated with ab228968 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

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

Perform heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

All lanes : Anti-Tissue Factor antibody [EPR22548-232] (ab228968) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate at 60 µg
Lane 2 : Tissue Factor knockout HAP1 whole cell lysate at 60 µg
Lane 3 : A431 (human epidermoid carcinoma epithelial cell), whole cell lysate at 20 µg
Lane 4 : MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 5 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 6 : BxPC-3 (human pancreas adenocarcinoma epithelial cell), whole cell lysate at 20 µg

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

Predicted band size: 33 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

ab228968 was shown to specifically react with Tissue Factor in wild-type HAP1 cells as signal was lost in Tissue Factor knockout cells. Wild-type and Tissue Factor knockout samples were subjected to SDS-PAGE. ab228968 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

The molecular weight observed is consistent with what has been described in the literature (PMID: 28938620).

Low expression: MCF-7 (PMID: 24137414, 28938620).

Exposure times: Lanes 1-2: 70 secs; Lanes 3-6: 3.25 secs.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human cervix carcinoma tissue labeling Tissue Factor with ab228968 at 1/500 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous and weak cytoplasmic staining on tumor cells of human cervix carcinoma (PMID: 26383146). The section was incubated with ab228968 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

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

Perform heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilizedA431 (human epidermoid carcinoma epithelial cell) and MCF7 () cells labeling Tissue Factor with ab228968 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in A431 cell line. Low expression: MCF7（PMID: 24137414, 28938620. The nuclear stain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

Flow cytometric analysis of MCF7 (human breast adenocarcinoma epithelial cell, Left) / A431 (human epidermoid carcinoma epithelial cell, Right) labeling Tissue Factor with ab228968 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Low expression: MCF7 (PMID: 24137414, PMID: 28938620).

Gated on viable cells.

Tissue Factor was immunoprecipitated from 0.35 mg A431 (human epidermoid carcinoma epithelial cell) whole cell lysate with ab228968 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab228968 at 1/1000 dilution. VeriBlot for IP for detection (HRP) (ab131366), was used Detection Reagent at 1/5000 dilution.

Lane 1: A431 whole cell lysate 10 µg (Input).
Lane 2: ab228968 IP in A431 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab228968 in A431 whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 30 seconds.