The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 10 µg/ml.
Use at an assay dependent concentration.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Detects a band of approximately 170, 350 kDa (predicted molecular weight: 177 kDa). The use of BSA as blocking agent is not recommended.
Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Required for brain and eye development. Promotes the disassembly of phosphorylated vimentin intermediate filaments (IF) during mitosis and may play a role in the trafficking and distribution of IF proteins and other cellular factors to daughter cells during progenitor cell division. Required for survival, renewal and mitogen-stimulated proliferation of neural progenitor cells.
CNS stem cells.
Belongs to the intermediate filament family.
Upon terminal neural differentiation, nestin is down-regulated and replaced by neurofilaments.
Constitutively phosphorylated. This increases during mitosis when the cytoplasmic intermediate filament network is reorganized.
Immunocytochemistry/ Immunofluorescence - Anti-Nestin antibody [Rat-401] (ab11306)This image is courtesy of an Abreview submitted by San San.
Immunocytochemical analysis of Rat neural stem cells from the hippocampus, labelling Nestin with ab11306. Samples were fixed in paraformaldehyde, permeablized in triton x-100 and blocked in 1% BSA for 40 minutes at room temperature. Incubation with ab11306 diluted 1/133 for 12 hours at 4°C. Secondary antibody was a polyclonal goat anti mouse FITC diluted 1/200.
Western blot - Anti-Nestin antibody [Rat-401] (ab11306)
Anti-Nestin antibody [Rat-401] (ab11306) at 1 µg/ml + MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg
Secondary Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/3000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 177 kDa
Exposure time : 20 minutes
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [Rat-401] (ab11306)This image is courtesy of an Abreview submitted by Mr Carl Hobbs
ab11306 staining Nestin in rat lateral ventricle (subventricular zone) sections by IHC-P (formaldehyde-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde and blocked with 1% BSA for 10 minutes at room temperature. Antigen retrieval was by heat mediation in citric acid (pH6). Samples were incubated with primary antibody 1/200 (TBS/BSA/azide pH7.5) for 24 hours. A Biotin-conjugated Goat polycolonal to mouse IgG, dilution 1/200, was used as secondary antibody. The image shows clear staining in cells of the rat subventricular zone of the lateral ventricle.
ICC/IF image of ab11306 stained PC12 cells. The cells were 100% methanol fixed (5 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 (ab11306, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 4% PFA fixed (10 min) PC12 cells at 10µg/ml.
IHC image of Nestin staining in 6 week old rat brain 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 ab11306, 1µg/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