SpecificitySpecies cross-reactivity includes human, rat, mouse, cow, and pig. These antibodies recognize epitopes in a small segment of the neurofilament NF-L subunit, which is not normally accessible to antibodies but becomes available upon 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.
Western blot (WB), Immunocytochemistry (ICC) / Immunofluorescence (IF). A dilution of 1:1,000 - 1:2,000 is recommended for WB. The antibody recognizes NF-L in reduced westerns regardless of the disease state.
Using standard fluorescent antibody methods, a 1:1,000 is recommended for ICC/IF/IH. 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. This antibody reacts to a peptide epitope within amino acids 311-375 of the human 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 antibodies recognize epitopes in a small segment of the neurofilament NF-L subunit, which is not normally accessible to antibodies but becomes available upon 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 HostRabbit
Antibody TypePolyclonal
Antibody IsotypeIgG
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 DescriptionAffinity 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 100 µL sterile-filtered, ultrapure water. 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™ rabbit antibody works well on western blots of a variety of species for NF-L protein detection, regardless of injury state, but binds only degenerated processes in sectioned material on injured or diseased brains. 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 section with a three-day-old midline C4 contusion injury by Immunofluorescence and Immunohistochemistry. The section was stained with R-2124-100, Neurofilament light polypeptide (NF-L) DG-Sensor™, Rabbit pAb, (red, 1:1,000) and co-stained with product M-1394-100, Neurofilament medium polypeptide (NF-M), Clone 3H11, Mouse mAb, (green, 1:1000). Product R-2124-100 does not stain the undamaged axons that M-1394-100 strongly stains (green). Linear arrays of swollen profiles originating from damaged axons, on the other hand, are strongly positive for R-2124-100, but not for the M-1394-100. In these regions, the M-1394-100 epitope, which is located in the C-terminal "tail" of NF-M, has been either removed or destroyed.
Analysis by western blot of NF-L expression in different tissue lysates with R-2124-100, Neurofilament light polypeptide (NF-L), DG-Sensor™, Rabbit pAb, (green, 1:2,000) with a band at approx 68-70kDa. Lane 1: protein standard (red); Lane 2: rat brain; Lane 3: rat spinal cord; Lane 4: mouse brain; Lane 5: mouse spinal cord; Lane 6: bovine spinal cord; Lane 7: pig spinal cord. DG-Sensor™ antibodies detect NF-L in denatured protein lysates regardless of degeneration state.