Alexa Fluor® 488 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated, Neurofilament H & M (NF-H/NF-M), Phospho, SMI 35

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Clone: SMI 35 (See other available formats)
Regulatory Status: RUO
Other Names: Neurofilament heavy polypeptide, NF-H, 200 kD neurofilament protein, neurofilament triplet H protein
Isotype: Mouse IgG1, κ
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SMI-35_A488_NeurofilamentH&M_Antibody_1_082718 — IHC staining of Alexa Fluor® 488 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated antibody (clone SMI 35) on formalin-fixed paraffin-embedded human cerebellum tissue. Following antigen retrieval using Sodium Citrate H.I.E.R. (Cat. No. 928602), the tissue was incubated with 5 µg/mL of the primary antibody overnight at 4°C. Nuclei were counterstained with DAPI, and the slides were mounted with ProLong™ Gold Antifade Mountant. The image was captured with a 40X objective. Scale Bar: 50 µm

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835613

25 µg

129 CHF

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835614

100 µg

323 CHF

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Description

Neurofilaments (NFs) are approximately 10 nanometer intermediate filaments found in neurons. They are a major component of the neuronal cytoskeleton, and function primarily to provide structural support for the axon and to regulate the axon diameter. There are three major NF subunits, and the names given to these subunits are based upon the apparent molecular mass of the mammalian subunits on SDS-PAGE. The light or lowest neurofilament (NF-L) runs at 68-70 kD. The medium or middle (NF-M) runs at about 145-160 kD, and the heavy or highest (NF-H) runs at 200-220 kD. However, the actual molecular weight of these proteins is considerably lower due to the highly charged C-terminal regions of the molecules. The level of NF gene expression correlates with the axonal diameter, which controls how fast electrical signals travel down the axon. Mutant mice with NF abnormalities have phenotypes resembling amyotrophic lateral sclerosis. NF immunostaining is common in diagnostic neuropathology. It is useful for differentiating neurons (positive for NF) from the glia (negative for NF).

Product Details

Technical Data Sheet (pdf)

Product Details

Verified Reactivity: Human, Mouse, Rat
Antibody Type: Monoclonal
Host Species: Mouse
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 488 under optimal conditions.
Concentration: 0.5 mg/ml
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: IHC-P - Quality tested

SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by formalin-fixed paraffin-embedded immunohistochemical staining. For immunohistochemistry, a concentration range of 5.0 - 10 µg/ml is suggested. It is recommended that the reagent be titrated for optimal performance for each application.

* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm.

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Excitation Laser: Blue Laser (488 nm)
Application Notes: Additional reported applications (for the relevant formats of this clone): include ELISA Capture^1-3, immunohistochemical staining on frozen tissue sections, immunofluorescence staining, and spatial biology (IBEX)^6,7.

Clone SMI 35 reacts with highly phosphorylated neurofilaments, as well as with low degrees of phosphorylation. It primarily reacts with neurofilament H and with neurofilament M to a lesser extent.

Notes: On two dimensional gels, this antibody detects a band extending from the phosphorylated neurofilament position at 200 kD (pI 5.1) toward the non-phosphorylated position at 170 kD (pI 6.2).
Additional Product Notes: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
RRID: AB_2750327 (BioLegend Cat. No. 835613)
AB_2750327 (BioLegend Cat. No. 835614)

Antigen Details

Structure: The medium or middle NF (NF-M) runs at about 145-160 kD, and the heavy or highest NF (NF-H) runs at 200-220 kD.
Distribution: Tissue Distribution: CNS, peripheral nerves and glandular cells of the prostate
Cellular Distribution: Cytoskeleton, nucleus, cytosol, and mitochondrion
Function: Neurofilaments are the major components of the neuronal cytoskeleton. They provide axonal support and regulate axon diameter.
Interaction: Cell bodies and dendrites are generally unstained. Other cells and tissues are unreactive except for peripheral axons.
Cell Type: Mature Neurons
Biology Area: Cell Biology, Neuroscience, Neuroscience Cell Markers
Molecular Family: Intermediate Filaments, Phospho-Proteins
Antigen References: Petzold A. 2012. Mult Scler Int. 2012:217802. PubMed

Gresle MM, et al. 2011. Mult Scler Int. 2011: 315406. PubMed
Gene ID: 4744 View all products for this Gene ID
UniProt: View information about Neurofilament HM NF-H NF-M Phospho on UniProt.org

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Related Protocols

Immunohistochemistry Protocol for Paraffin-Embedded Sections

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Related FAQs

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Are other fluorophores compatible with IBEX?: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
The same antibody works in one tissue type but not another. What is happening?: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
How can I be sure the staining I’m seeing in my tissue is real?: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.

Other Formats

View All Neurofilament H&M (NF-H/NF-M), Phospho Reagents Request Custom Conjugation

Description	Clone	Applications
Anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated	SMI 35	IHC-P,WB,IHC-F,ELISA
Purified anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated	SMI 35	IHC-P,WB,IHC-F
Biotin anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated	SMI 35	WB
Alexa Fluor® 594 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated	SMI 35	IHC-P,ICC
Alexa Fluor® 647 anti NF-H/NF-M, Hypophosphorylated	SMI 35	IHC-P
Alexa Fluor® 488 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated	SMI 35	IHC-P,SB

Certificate of Analysis

Go To Top Version: 2 Revision Date: 04.22.2022

For Research Use Only. Not for diagnostic or therapeutic use.

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

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