Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

This data was developed using ab268020, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human esophageal squamus cell carcinoma labelling fibronectin with ab268020 at a concentration of 0.05µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab268020 anti-fibronectin antibody [EPR23110-46] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with ab268020 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cells is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of paraffin-embedded human esophageal squamous cell carcinoma tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the stroma of human esophageal squamous cell carcinoma (PMID : 30314454). Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the stroma of human mammary gland (PMID : 650101). Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of paraffin-embedded Human pancreatic carcinoma tissue labeling Fibronectin with ab268020 at 1/4000 dilution (0.146 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). The section was incubated with ab268020 at 4°C overnight. Positive staining in the human pancreatic carcinoma counterstained with hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP)(ab214880) Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat colon tissue labeling Fibronectin with ab268020 at 1/100 dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Positive staining mainly on smooth muscle of rat colon (PMID : 17881565) is observed. The nuclear counterstain is DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the smooth muscle of mouse stomach (PMID : 650101) Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the smooth muscle of rat stomach (PMID : 650101) Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Fibronectin with ab268020 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cells is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse colon tissue labeling Fibronectin with ab268020 at 1/100 dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Positive staining mainly on smooth muscle of mouse colon (PMID : 17881565) is observed. The nuclear counterstain is DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

• WB

Lab

Western blot - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

This data was developed using the same antibody clone in a different buffer formulation (ab268020).

Western blot : Rabbit Monoclonal[EPR23110-46] to Fibronectin ab268020 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 238-268 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in FN1 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Fibronectin antibody [EPR23110-46] (<a href='/en-us/products/primary-antibodies/fibronectin-antibody-epr23110-46-ab268020'>ab268020</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human FN1 knockout MCF7 cell line (ab286324) at 20 µg

Lane 3:

HepG2 at 20 µg

Lane 4:

PC-3 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 272 kDa

Observed band size: 238 kDa,268 kDa

false

• WB

Lab

Western blot - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (AB268022)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab268020).

ab268020 was shown to specifically react with Fibronectin in wild-type HAP1 cells as signal was lost in Fibronectin knockout cells. Wild-type and Fibronectin knockout samples were subjected to SDS-PAGE. ab268020 and ab129002 (Rabbit anti-Vinculin loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/5000 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.

Exposure time : Lanes 1-2 : 37 seconds; Lanes 3-4 : 3 minutes.

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

All lanes:

Western blot - Anti-Fibronectin antibody [EPR23110-46] - BSA and Azide free (ab268022) at 0.585 µg/mL

Lane 1:

Human stomach tissue lysate at 10 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg

Lane 3:

Fibronectin-1 knockout HAP1 whole cell lysate at 40 µg

Lane 4:

Wild-type HAP1 whole cell lysate at 40 µg

Predicted band size: 262 kDa

Observed band size: 285 kDa

true