The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 59 kDa (predicted molecular weight: 62 kDa). Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.
Use at an assay dependent concentration.
FunctionFunctional ligand of integrin alpha-8/beta-1 in kidney development. Regulates the expression of GDNF with integrin alpha-8/beta-1 which is essential for kidney development. May also play a role in the development and function of various tissues, regulating cell adhesion, spreading and survival through the binding of several integrins.
Tissue specificityExpressed in kidney and lung and to a lower extent in brain, pregnant uterus, placenta, thyroid gland and blood vessels.
Sequence similaritiesBelongs to the nephronectin family. Contains 5 EGF-like domains. Contains 1 MAM domain.
Developmental stageExpressed in fetal kidney, cochlea, eye, heart and lung.
DomainThe MAM domain is required for localization at the cell surface.
Cellular localizationSecreted > extracellular space > extracellular matrix. Trapped on the cell surface or in the extracellular matrix.
ICC/IF image of ab64419 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64419, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
IHC image of ab64419 staining in Human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab64419, 5µg/ml, for 15 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
References for Anti-nephronectin antibody (ab64419)
This product has been referenced in:
Lennon R et al. Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix. J Am Soc NephrolN/A:N/A (2014).
Read more (PubMed: 24436468) »