SpecificitySpecies cross-reactivity includes human, rat, mouse, cow, and pig. These are antibodies that recognize epitopes in a small segment of the neurofilament NF-L subunit, which are normally not accessible to antibodies but which became available on degeneration in natural or native conditions. Under reduced or proteinase-treated tissues or western blot, these antibodies are all specific for NF-L from a variety of species
Species ReactivityBovine, Human, Mouse, Pig, Rat
Immunogen DescriptionThe antibody has been made against a proprietary recombinant construct containing amino acids of human NF-L expressed in and purified from E Coli.
A dilution of 1:5,000 - 1:10,000 is recommended for WB. The antibody recognizes NF-L in reduced westerns regardless of the disease state.
Using standard fluorescent antibody methods, a 1: 5000-10,000 dilution is recommended for ICC/IF. For example, block and permeabilize sections in 5-10% normal goat serum or serum of the species the secondary antibodies were made in, in PBS plus 1% Triton-100 (PBST) for 1 hour with slight agitation, followed by primary antibody incubations and fluorescent secondary identification. High primary antibody dilutions require refrigerated, overnight incubations for best results. Recommended fixation is 4% PFA fixed, frozen tissue 20-50 microns; other fixation methods have not been tested and are not recommended at this time.
Degeneration-specific detection is fixation and antigen recovery dependent. The epitope detected is the peptide LLKAK within NF-L. This peptide epitope is uncovered in degenerating cells but not normal cells. However, the specific reactivity of this epitope is sensitive to tissue treatments and could become exposed in healthy cells under some conditions. For example, treatment of the fixed tissue with high temperatures, proteinase, or other denaturants may cause the reactive epitope to become exposed in healthy cells, leading to a false positive. Biosensis recommends experimenting with treated and untreated tissues when first using these antibodies if degeneration specificity is desired. The exact conditions and dilutions must be determined experimentally by the end user.
This antibody will detect NL-L protein in paraffin-embedded tissues; however, the degeneration-specific detection can be problematic in paraffin tissues, particularly if Heat-Induced Epitope Retrieval (HIER), or other common antigen recovery methods are used. This is because the reactive epitope, which is covered in healthy cells but exposed in degenerative cells, could become accessible to the antibody in healthy cells, leading to false positives. For this reason, paraffin-embedded tissues are not recommended if degeneration-specific detection is desired.
SpecificitySpecies cross-reactivity includes human, rat, mouse, cow, and pig. These are antibodies that recognize epitopes in a small segment of the neurofilament NF-L subunit, which are normally not accessible to antibodies but which became available on degeneration in natural or native conditions. Under reduced or proteinase-treated tissues or western blot, these antibodies are all specific for NF-L from a variety of species
Target Host SpeciesHuman
Species ReactivityBovine, Human, Mouse, Pig, Rat
Antibody HostMouse
Antibody TypeMonoclonal
Antibody IsotypeIgG1
Clone Name6H63
ConjugateUnconjugated
Immunogen DescriptionThe antibody has been made against a proprietary recombinant construct containing amino acids of human NF-L expressed in and purified from E Coli.
Purity DescriptionProtein G purified
FormatLyophilized from PBS buffer pH 7.2-7.6 with 0.1% trehalose, and sodium azide
Reconstitution InstructionsSpin vial briefly before opening. Reconstitute with 50 µL sterile-filtered, ultrapure water to achieve a 1 mg/mL concentration. Centrifuge to remove any insoluble material.
Storage InstructionsStore lyophilized antibody at 2-8°C After reconstitution of lyophilized antibody, aliquot and store at -20°C for a higher stability. Avoid freeze-thaw cycles. Store at 4°C for up to one month for short term storage and frequent use.
Batch NumberPlease see item label.
Expiration Date12 months after date of receipt (unopened vial).
Scientific BackgroundOur DG-Sensor™ clone 6H63 binds only degenerating or degenerated neuronal processes. Its immunogen is a proprietary recombinant immunogen based on the Coil 2 region of human NF-L. In addition, the antibody works well on denatured western blots of various species for NF-L, regardless of inquiry. The antibody works in both paraffin and 4% PFA fixed tissue. Caution: epitope reactivity is sensitive to protease attack; proteinase antigen recovery is not recommended and could lead to false positives in staining.
Image of a rat spinal cord coronal section with a three-day-old midline C4 contusion injury by Immunofluorescence and Immunohistochemistry. The section was stained with M-2122-50, Neurofilament light polypeptide (NF-L), DG-Sensor™, Clone 6H63, Mouse mAb, (green), and co-stained with product R-2113-50, Neurofilament light polypeptide, C-terminus, (NF-L-Ct), Rabbit pAb, (red). Product M-2122-50 stains prominent material aggregates in the lateral funiculi and dorsal columns, where the material is concentrated but less so elsewhere in the section. Due to the C4 lesion, these axons are deteriorating and dying. As product R-2113-50 binds to the C-terminal "tail" region of NF-L, which is absent or destroyed during degeneration, only M-2122-50 staining is observed in the degenerating neurons; positive staining for R-2113-50 in those regions is largely negative.
Image of formalin-fixed paraffin-embedded sections of a mouse model of ALS (Acta Neuropathol. 2016 Jan; 131(1): 103-114) by Immunohistochemistry. The section was stained with M-2122-50, Neurofilament light polypeptide (NF-L), DG-Sensor™, Clone 6H63, Mouse mAb. These heterozygous G85R-SOD1-YFP mice do not develop ALS unless primed with mutant SOD1 aggregates, indicating that this is a model of prion induction of pathogenesis. Degenerated processes were seen in the sciatic nerve, spinal cord fibers, and, as shown here, the brain stem, after this mouse was injected with mutant SOD1. Take note of the swollen and sinusoidal profiles. The inset depicts a beaded profile typical of degenerating processes.
Analysis by western blot of NF-L expression in crude CNS homogenates with M-2122-50, Neurofilament light (NF-L), DG-Sensor™ Clone 6H63, Mouse mAb, (green, 1:5,000) with a band at approx 68-70kDa. Lane 1: molecular weight standards of the specified size (red); Lane 2: E20 rat spinal cord homogenate; Lane 3: adult rat spinal cord homogenate; Lane 4: bovine spinal cord extract homogenate. Product M-2122-50 recognizes denatured forms of NF-L with apparent molecular weights of 68-70kDa when immobilized on western blot membranes. Lower molecular weight bands beneath the major band are NF-L proteolytic fragments.