Anti-Neurofilament 200 kDa Antibody, clone NE14

Neurofilaments are a type of intermediate filament that serve as major elements of the cytoskeleton supporting the axon cytoplasm. They are the most abundant fibrillar components of the axon, being on average 3-10 times more frequent than axonal microtubules. Neurofilaments (10nm in dia.) are built from three intertwined protofibrils which are themselves composed of two tetrameric protofilament complexs of monomeric proteins. The neurofilament triplet proteins (68/70, 160, and 200 kDa) occur in both the central and peripheral nervous system and are usually neuron specific. The 68/70 kDa NF-L protein can self-assemble into a filamentous structure, however the 160 kDa NF-M and 200 kDa NF-H proteins require the presence of the 68/70 kDa NF-L protein to co-assemble. Neuromas, ganglioneuromas, gangliogliomas, ganglioneuroblastomas and neuroblastomas stain positively for neurofilaments. Although typically restricted to neurons, neurofilaments have been detected in paragangliomas and adrenal and extra-adrenal pheochromocytomas. Carcinoids, neuroendocrine carcinomas of the skin, and oat cell carcinomas of the lung also express neurofilaments. For more neurofilament information see Nervous System Cell Type Specific Marker chart online under the CHEMICON Technical Support section.

The antibody reacts with neurofilament 200 kD.

Purified neurofilament polypeptides (Debus et al., 1983).

Detect Neurofilament 200 kDa using this Anti-Neurofilament 200 kDa Antibody, clone NE14 validated for use in IH.

Immunohistochemistry: 5-10 μg/mL (See below protocol.)

Optimal working dilutions must be determined by end user.

Immunohistochemistry Protocol for Anti-Neurofilament 200 kD

Ideal specimens are obtained from frozen sections from shock-frozen tissue samples. The frozen sections are dried in the air and then fixed with acetone at -15 to -25°C for 10 min. Excess acetone is allowed to evaporate at 15-25°C. Material fixed in alcohol and embedded in paraffin can also be used, see (Altmannsberger et al., 1982). The antibody appears to react with tissue fixed in formaldehyde for a short time (10 min) (Debus et al., 1983). Other fixation conditions must be first tested by the investigator.

It is advantageous to block unspecific binding sites by overlaying the sections with fetal calf serum for 20-30 min at 15-25°C. Excess of fetal calf serum is removed by decanting before application of the antibody solution. Cytocentrifuge preparations of single cells or cell smears are also fixed in acetone. These preparations should, however, not be dried in the air. Instead, the excess acetone is removed by briefly washing in phosphate-buffered saline (PBS).

Further treatment is then as follows:

Overlay the preparation with 10-20 μL antibody solution and incubate in a humid chamber at 37°C for 1 h.

Dip the slide briefly in PBS and then wash 3 times in PBS for 3 min (use fresh PBS each time)

Wipe the margins of the preparation dry and overlay the preparation with 10-20 μL of an anti-mouse Ig-FITC or anti-mouse Ig-POD antibody and allow to incubate for 1 h at 37°C in a humid chamber.

Wash the slide as described above.

The preparation must not be allowed to dry out during any of the steps.

If using an indirect immunofluorescence technique, the preparation should be overlaid with a suitable embedding medium (e.g. Moviol, Hoechst) and examined under the fluorescence microscope. If a POD-conjugate has been used as the secondary antibody, the preparation should be overlaid with a substrate solution (see below) and incubated at 15-25°C until a clearly visible red-brown color develops. A negative control (e.g. only the secondary antibody) should remain unchanged in color during this incubation period. Subsequently, the substrate is washed off with PBS and the preparation is stained, if desired, with hemalum stain for about 1 min. The hemalum solution is washed off with PBS, the preparation is embedded and examined.

Substrate solutions:

Aminoethyl-carbazole:

Dissolve 2 mg 3-amino-9-ethylcarbazole with 1.2 mL dimethylsulfoxide and add 28.8 mL 0.05 M Tris-HCl, pH 7.3, and 20 μL 3% H 2 O 2 (w/v). Prepare solution freshly each day.

Diaminobenzidine:

Dissolve 25 mg 3,3′-diaminobenzidine with 50 mL 0.05 M Tris-HCl, pH 7.3, and add 40 μL 3% H2O2 (w/v). Prepare solution freshly each day.

Research Category
Neuroscience

Research Sub Category
Neurofilament & Neuron Metabolism

Neuronal & Glial Markers

Format: Purified

Purified immunoglobulin. Liquid. Buffer = 0.02M Phosphate buffer, 0.25M NaCl containing 0.1% sodium azide.

Maintain refrigerated at 2-8°C in undiluted aliquots for up to 6 months.DO NOT FREEZE

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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