Mixed neuron/glial cultures stained with mouse monoclonal antibody to neurofilament subunit NF-L MCA-7D1
(green) and CPCA-NF-H, EnCor’s rabbit antibody to neurofilament NF-H. 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.
Neurofilaments can be defined as the intermediate or 10nm filaments found in specifically in neuronal cells. In the electron microscope neurofilaments appears as 10nm diameter fibers of indeterminate length which generally have fine wispy protrusions from their sides. They are particularly abundan in axons of large projection neurons. They probably function to provide structural support for neurons and their synapses and to support the large axon diameters required for rapid conduction of impulses down axons. They are composed a mixture of subunits which usually include the three neurofilament triplet proteins, known as NF-L, NF-M and NF-H. Neurofilaments may also include smaller amounts of peripherin, alpha-internexin, nestin and in some cases vimentin.
Antibodies to the various neurofilament subunits are very useful cell type markers since the proteins are among the most abundant in the nervous system, are expressed only in neurons, and are biochemically very stable. The characterization of this antibody has been published in peer-reviewed form and has been shown to be very effective in ELISA (1).
The antibody has a very high titer and can be used in immunofluoresence experiments at dilutions of 1 to 1 million, and reacts primarily with the phosphorylated axonal form of NF-H. As with many antibodies to NF-H, this antibody has some cross-reactivity with phosphorylated NF-M, which has similar phosphorylation sites to NF-H.
To raise this particular antibody bovine 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 (2). 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. The HGNC name for this protein is NEFH.
This antibody was generated in chicken by standard procedures and immunoglobulin was extracted from egg yolk. The resulting polyclonal antibody belongs to the IgY subclass. This is the chicken homologue of mammalian IgG and can be used in the same general way, with the caveat that this type of antibody does not bind either Protein A or Protein G. Suitable second antibody reagents can be obtained from many vendors including Molecular Probes and Sigma-Aldrich.
For some on line images generated with this antibody press here
1. Shaw G, Yang C, Ellis R, Anderson K, Parker Mickle J, Scheff S, Pike B, Anderson DK and Howland DR. Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury. Biochem Biophys Res Commun. 336:1268-1277 (2005).
2. Delacourte A, Filliatreau G, Boutteau F, Biserte G, Schrevel J. Study of the 10-nm- filament fraction isolated during the standard microtubule preparation. Biochem J. 191:543-6 (1980).