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Full length native protein (cow intermediate filaments were prepared from spinal cords by the glycerol polymerization method of Delacourte et al., and the cytoskeletal material was dissolved in 6M urea. Individual neurofilament subunits were purified by ion exchange chromatography on DEAE-cellulose and the NF-H containing fractions were concentrated and further purified by preparative gel electrophoresis on a Biorad Prepcell).
Our Abpromise guarantee covers the use of ab4680 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC - Wholemount||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration. PubMed: 19944698|
|WB||1/1e+006. Predicted molecular weight: 200-220 kDa.|
Immunohistochemical staining of Neurofilament heavy polypeptide in aplysia buccal ganglia with ab4680 at 1/1000. The sample was permeabilized with 2% Triton X100 and blocked with 1%BSA/5% goat serum for 2 hours at room temperature, before being incubated with the primary antibody for 1 hour at room temperature in 5% goat serum. ab96949 (goat anti-chicken IgY H&L DyLight® 594) was used as the secondary antibody at 1/200.
Mixed neuron/glial cultures stained with a mouse monoclonal antibody to neurofilament subunit NF-L (green) and ab4680, chicken antibody to neurofilament heavy polypeptide. This antibody binds primarily to the phosphorylated axonal forms of NF-H, in contrast to the NF-L antibody which stains both axonal and dendritic/perikaryal neurofilaments. The NF-L antibody therefore reveals a prominent cell body in green, while the surrounding axonal profiles are orange, since the are bound by both NF-L and the chicken NF-H antibody. Blue is a DNA stain.
Rat Neurons stained with chicken anti-NF-H (green). The NF-H antibody was used at a dilution of 1/100000 using our standard fixation and staining procedure (in protocol section).
ab4680 at 1/10000 dilution staining Neurofilament heavy polypeptide in rat skin tissue by immunohistochemistry (frozen sections). Sections were fixed using Bouin's Fixative and permeabilized in 0.1% Triton X-100 prior to blocking in 10% serum for 1 hour at 24°C and then incubated with ab4680 for 24 hours at 4°C. A donkey polyclonal to chicken IgY, diluted 1/100 was used as the secondary antibody.
ab4680 staining Neurofilament heavy polypeptide (green) in murine embryoid bodies by Immunocytochemistry/ Immunofluorescence. Nuclei were visualized with DAPI.
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