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).

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).

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).

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).

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.

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).

All lanes : Anti-Fibronectin antibody [EPR23110-46] (ab268020) at 0.585 µg/ml (1:1000 dilution)

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

Developed using the ECL technique.

Predicted band size: 262 kDa
Observed band size: 285 kDa why is the actual band size different from the predicted?

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 : Anti-Fibronectin antibody [EPR23110-46] (ab268020) at 1/1000 dilution

Lane 1 : Human serum
Lane 2 : Human plasma
Lane 3 : Human kidney tissue lysate
Lane 4 : Human stomach tissue lysate
Lane 5 : Mouse plasma
Lane 6 : Rat plasma

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-4 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 5-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 262 kDa
Observed band size: 258 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-2, 5-6: 3 seconds; Lane 3: 125 seconds; Lane 4: 33 seconds.

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

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).

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.