重组Anti-N Cadherin抗体[EPR1791-4] (ab76011)

Immunohistochemical analysis of formalin-fixed paraffin-embedded human heart labelling N Cadherin with ab271856 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab271856 anti N Cadherin antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation (ab271856).

False colour image of Western blot: Anti-N Cadherin antibody [EPR1791-4] staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab76011 was shown to bind specifically to N Cadherin. A band was observed at 125 kDa in wild-type HeLa cell lysates with no signal observed at this size in cdh2 knockout cell line ab274934 (knockout cell lysate ab274992).

To generate this image, wild-type and cdh2 knockout HeLa cell lysates were analysed. First, 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 3% milk before ab76011 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cardiac muscle tissue sections labeling N Cadherin with purified ab76011 at 1:50 dilution (1.94 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Lanes 1 - 2: Merged signal (red and green). Green - ab76011 observed at 125 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab76011 was shown to react with N Cadherin in wild-type HEK-293T. Loss of signal was observed when knockout cell line ab255377 (knockout cell lysate ab263843) was used. Wild-type and N Cadherin knockout samples were subjected to SDS-PAGE. ab76011 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

The molecular weight observed is consistent with what has been described in the literature (PMID: 22553038). This antibody fails to detect N Cadherin in HCT 116 cell which is positive described in the literature (PMID: 23431386 and 26540342)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling N Cadherin with purified ab76011 at 1:50 dilution (1.94 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry of kidney carcinoma staining N Cadherin with ab76011 at 1μg/ml

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab76011 staining N Cadherin in Mouse pancreas tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA + 1% FBS for 2 hours at room temperature; antigen retrieval was by heat mediation in a citrate buffer pH6. Samples were incubated with primary antibody (1/500 in 1% BSA + 1% FBS) for 16 hours at 4°C. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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