MAB5256
Anti-Neurofilament 200 kDa Antibody, clone NE14
clone NE14, Chemicon®, from mouse
Sinonimo/i:
Anti-CMT2CC, Anti-NFH
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About This Item
Codice UNSPSC:
12352203
eCl@ss:
32160702
NACRES:
NA.41
Prodotti consigliati
Origine biologica
mouse
Livello qualitativo
Forma dell’anticorpo
purified immunoglobulin
Tipo di anticorpo
primary antibodies
Clone
NE14, monoclonal
Reattività contro le specie
rat, pig, human
Produttore/marchio commerciale
Chemicon®
tecniche
immunohistochemistry: suitable
Isotipo
IgG1
N° accesso NCBI
N° accesso UniProt
Condizioni di spedizione
wet ice
modifica post-traduzionali bersaglio
unmodified
Informazioni sul gene
human ... NEFH(4744)
pig ... Nefh(100156492)
rat ... Nefh(24587)
Descrizione generale
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.
Specificità
The antibody reacts with neurofilament 200 kD.
Immunogeno
Purified neurofilament polypeptides (Debus et al., 1983).
Applicazioni
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.
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
Neuroscience
Research Sub Category
Neurofilament & Neuron Metabolism
Neuronal & Glial Markers
Neurofilament & Neuron Metabolism
Neuronal & Glial Markers
Stato fisico
Format: Purified
Purified immunoglobulin. Liquid. Buffer = 0.02M Phosphate buffer, 0.25M NaCl containing 0.1% sodium azide.
Stoccaggio e stabilità
Maintain refrigerated at 2-8°C in undiluted aliquots for up to 6 months.DO NOT FREEZE
Altre note
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
Note legali
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Esclusione di responsabilità
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
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Codice della classe di stoccaggio
10 - Combustible liquids
Classe di pericolosità dell'acqua (WGK)
WGK 2
Punto d’infiammabilità (°F)
Not applicable
Punto d’infiammabilità (°C)
Not applicable
Certificati d'analisi (COA)
Cerca il Certificati d'analisi (COA) digitando il numero di lotto/batch corrispondente. I numeri di lotto o di batch sono stampati sull'etichetta dei prodotti dopo la parola ‘Lotto’ o ‘Batch’.
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