The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml.
Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. NF-H has an important function in mature axons that is not subserved by the two smaller NF proteins.
Involvement in disease
Defects in NEFH are a cause of susceptibility to amyotrophic lateral sclerosis (ALS) [MIM:105400]. ALS is a neurodegenerative disorder affecting upper and lower motor neurons, and resulting in fatal paralysis. Sensory abnormalities are absent. Death usually occurs within 2 to 5 years. The etiology is likely to be multifactorial, involving both genetic and environmental factors.
Belongs to the intermediate filament family.
There are a number of repeats of the tripeptide K-S-P, NFH is phosphorylated on a number of the serines in this motif. It is thought that phosphorylation of NFH results in the formation of interfilament cross bridges that are important in the maintenance of axonal caliber. Phosphorylation seems to play a major role in the functioning of the larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincident with a change in the neurofilament function. Phosphorylated in the Head and Rod regions by the PKC kinase PKN1, leading to inhibit polymerization.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurofilament heavy polypeptide antibody (ab105453)
IHC image of Neurofilament heavy polypeptide staining in human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab105453, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
Immunocytochemistry/ Immunofluorescence - Anti-Neurofilament heavy polypeptide antibody (ab105453)
ICC/IF image of ab105453 stained SHSY5Y cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab105453, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) SHSY5Y cells at 5µg/ml.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Neurofilament heavy polypeptide antibody (ab105453)This image is courtesy of an abreview submitted by Sophie Pezet, Laboratoire de Neurobiologie, France
IHC-FoFr image of Neurofilament heavy polypeptide staining on rat hippocampus sections using ab105453 (/100).The sections used came from animals perfused fixed with Paraformaldehyde 4% with 15% of a solution of saturated picric acid, in phosphate buffer 0.1M. Following postfixation in the same fixative overnight, the spinal cord were cryoprotected in sucrose 30% overnight. Spinal cords were then cut using a cryostat and the immunostainings were performed using the ‘free floating’ technique.