Anti-Fibronectin antibody [EP-5]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EP-5] (AB281574)

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling Fibronectin with ab281574 at 1/100 (10.52 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in stromal cells of human colon cancer. The section was incubated with ab281574 for 30 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 LeicaDS9800 (Bond® Polymer Refine Detection) .

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EP-5] (AB281574)

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling Fibronectin with ab281574 at 1/100 (10.52 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in stromal cells of human cervical cancer. The section was incubated with ab281574 for 30 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 LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EP-5] (AB281574)

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling Fibronectin with ab281574 at 1/100 (10.52 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in stromal cells of human prostatic hyperplasia. The section was incubated with ab281574 for 30 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 LeicaDS9800 (Bond® Polymer Refine Detection) .

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EP-5] (AB281574)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-87 MG cells labelling Fibronectin with ab281574 at 1/50 (21.04 ug/ml) dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and membranous staining in U-87 MG cell line. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• WB

Lab

Western blot - Anti-Fibronectin antibody [EP-5] (AB281574)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : Lane 1 : 125 seconds
Lane 2 : 70 seconds

All lanes:

Western blot - Anti-Fibronectin antibody [EP-5] (ab281574) at 1/1000 dilution

Lane 1:

Human colon lysate at 10 µg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 262 kDa

Observed band size: 285 kDa

false

• WB

Lab

Western blot - Anti-Fibronectin antibody [EP-5] (AB281574)

Blocking and diluting buffer and concentration : ntercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 28367057).

Lanes 1-3 : Merged signal (red and green). Green - ab281574 observed at 285kDa. Red - loading control ab52901(Rabbit monoclonal [EP1331Y] to beta Tubulin) observed at 50 kDa.

Lanes 1-2 : ab281574 Anti-Fibronectin antibody was shown to react with Fibronectin in HAP1 cells in Western blot. Loss of signal was observed when Fibronectin knockout sample was used. Wild-type and Fibronectin knockout samples were subjected to SDS-PAGE.

ab281574 and Anti-beta Tubulin antibody [EP1331Y] - Loading Control (ab52901) were incubated at 4℃ overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) preadsorbed (ab216772) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Fibronectin antibody [EP-5] (ab281574) at 1/500 dilution

Lane 1:

Wild type HAP1(human chronic myelogenous leukemia near-haploid cell line), whole cell lysate at 20 µg

Lane 2:

Fibronectin knockout HAP1(human chronic myelogenous leukemia near-haploid cell line), whole cell lysate at 20 µg

Lane 3:

Human plasma lysate at 20 µg

Secondary

All lanes:

Goat Anti-Mouse IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 262 kDa

Observed band size: 285 kDa

false