The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 2 µg/ml.
A diffuse signal is seen throughout the cells if higher concentrations are used (5-10µg/ml). We have had reports that the antibody works less well in this application in murine (3T3) cells.
Use a concentration of 2 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 135 kDa (predicted molecular weight: 100 kDa).
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Cadherins are members of a multigene family of single chain glycoprotein receptors mediating calcium dependent cell-cell adhesion. They play an important role in the growth and development of cells via the mechanisms of control of tissue architecture and the maintenance of tissue integrity. Cadherins are expressed in a tissue specific manner and and are required for assembly of cells into solid tissue. Individual cadherin molecules are known to co-operate with each other to form a linear cell adhesion zipper. In adhesion junctions cadherins are bound to beta and gamma catenins which in turn bind to alpha catenin, an actin binding protein. Cadherins play an important part in tumor invasion and metastasis.
ICC/IF image of ab16505 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16505, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
IHC image of pan Cadherin staining in human liver FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16505, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
Western blot - Anti-pan Cadherin antibody (ab16505)
All lanes : Anti-pan Cadherin antibody (ab16505) at 1 µg/ml
Lane 1 : Mouse heart Lane 2 : HeLa cell lysate Lane 3 : 3T3 cell lysate Lane 4 : Mouse muscle Lane 5 : Human heart Lane 6 : Mouse heart with Human pan Cadherin peptide (ab17098) at 1 µg/ml Lane 7 : HeLa cell lysate with Human pan Cadherin peptide (ab17098) at 1 µg/ml Lane 8 : 3T3 cell lysate with Human pan Cadherin peptide (ab17098) at 1 µg/ml Lane 9 : Mouse muscle with Human pan Cadherin peptide (ab17098) at 1 µg/ml Lane 10 : Human heart with Human pan Cadherin peptide (ab17098) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Secondary Goat anti-rabbit conjugated to Alexafluor 680 at 1/10000 dilution
Performed under reducing conditions.
Predicted band size : 100 kDa
Immunocytochemistry - Anti-pan Cadherin antibody (ab16505)This image is courtesy of Rosmaria Mangiacasale & Patrizia Lavia, University La Sapienza
HeLa cells fixed in methanol and stained with ab16505 (2µg/ml). The cells were fixed in 100% methanol for 6 minutes at -20°C, then washed once in PBS. The 2 images show the cells stained with different secondary antibodies, Donkey anti Rabbit FITC (image A) and Donkey anti Rabbit Cy3 (image B). In each case ab16505 stains the plasma membrane. In image A ab16505 is stained green and in image B ab16505 is stained red. In both images the DNA is stained with DAPI (blue).
Immunocytochemistry/ Immunofluorescence - Anti-pan Cadherin antibody (ab16505)Image from Kiss K et al., PLoS One. 2012;7(5):e37378. Epub 2012 May 24. Fig 1.; doi:10.1371/journal.pone.0037378; May 24, 2012, PLoS ONE 7(5): e37378.
Immunofluorescence analysis of HeLa cells, staining pan Cadherin (red) with ab16505.
Cells were fixed with paraformaldehyde, permeabilized in methanol and blocked for 1 hour at room temperature in DPBS containing 2 mg/mL BSA, 1% fish gelatin, 0.1% Triton-X 100 and 5% goat serum. Cells were then incubated for 1 hour at room temperature with the primary antibody diluted in blocking buffer.
An AlexaFluor®-conjugated anti-rabbit IgG was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).