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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 fibres of indeterminate length which generally have fine wispy protrusions from their sides. They are found particularly abundantly in axons of large projection neurons. Antibodies to the various neurofilament subunits are very useful cell type markers since the proteins are among the most abundant of the nervous system, are expressed only in neurons, and are biochemically very stable.
Images: Mixed neuron/glial rat brain cultures stained with RPCA-NF-H-pind (green) and chicken polyclonal antibody to Glial Fibrillary Acidic Protein-GFAP (red). The neurofilament NF-H antibody binds to phosphorylated axonal forms of NF-H and non-phosphorylated dendritic and perikaryal forms. Protocol on datasheet.
Mammalian NF-H contains tandem repeats based on the sequence lysine serine proline (KSP), which may be present up to 60 times, with some variability between species. These KSP sequences are the sites of phosphorylation, and in axons most if not all of the serine residues are phosphorylated. In contrast, neurofilaments in dendrites and perikarya are not phosphorylated on these KSP sequences. Axonal neurofilaments are therefore quite different immunologically from those in perikarya and dendrites, and many antibodies stain only axonal or only dendritic/perikaryal neurofilaments. Since bacteria do not phosphorylate mammalian proteins, this protein resembles the NF-H form characteristic of perikaryal and dendritic neurofilaments. Accordingly this antibody stains dendritic and perikaryal neurofilaments particularly well.
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes by Category
Image: Blot of rat spinal cord extract blotted with NF-H, phosphate independent. A major band running at 200kDa is NF-H, the major neurofilament subunit protein. A minor band at about 160kDa is the non-phosphorylated dendritic and perilaryal form of this protein. Protocol on datasheet.