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Rabbit Recombinant Monoclonal NFM antibody. Suitable for ICC/IF, IP, Flow Cyt (Intra), ELISA, WB, IHC-Fr, IHC-P and reacts with Mouse, Rat, Human samples. Cited in 2 publications.

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Images

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348), expandable thumbnail
  • Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348), expandable thumbnail
  • Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348), expandable thumbnail

Publications

Key facts

Isotype
IgG
Host species
Rabbit
Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form
Liquid
Clonality
Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
ICC/IFIPFlow Cyt (Intra)ELISAWBIHC-FrIHC-P
Human
Tested
Expected
Tested
Expected
Tested
Expected
Tested
Mouse
Tested
Tested
Tested
Tested
Tested
Tested
Tested
Rat
Tested
Tested
Tested
Expected
Tested
Tested
Tested

Tested
Tested

Species
Mouse
Dilution info
1/50
Notes

-

Species
Rat
Dilution info
1/50
Notes

-

Species
Human
Dilution info
1/50
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species
Mouse
Dilution info
1/30
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Species
Rat
Dilution info
1/30
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species
Human
Dilution info
Use at an assay dependent concentration.
Notes

-

Tested
Tested

Species
Mouse
Dilution info
1/500
Notes

-

Species
Rat
Dilution info
1/500
Notes

-

Species
Human
Dilution info
1/500
Notes

-

Tested
Tested

Species
Mouse
Dilution info
0.125 µg/mL
Notes

-

Expected
Expected

Species
Rat, Human
Dilution info
Use at an assay dependent concentration.
Notes

-

Tested
Tested

Species
Mouse
Dilution info
1/1000
Notes

-

Species
Rat
Dilution info
1/1000
Notes

-

Species
Human
Dilution info
1/1000
Notes

-

Tested
Tested

Species
Mouse
Dilution info
1/100
Notes

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

Species
Rat
Dilution info
1/100
Notes

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

Expected
Expected

Species
Human
Dilution info
Use at an assay dependent concentration.
Notes

-

Tested
Tested

Species
Mouse
Dilution info
1/4000
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Species
Rat
Dilution info
1/4000
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Species
Human
Dilution info
1/4000
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target data

Function

Neurofilaments usually contain three intermediate filament proteins: NEFL, NEFM, and NEFH which are involved in the maintenance of neuronal caliber. May additionally cooperate with the neuronal intermediate filament proteins PRPH and INA to form neuronal filamentous networks (By similarity).

Alternative names

NEF3, NFM, NEFM, Neurofilament medium polypeptide, NF-M, 160 kDa neurofilament protein, Neurofilament 3, Neurofilament triplet M protein

Recommended products

Rabbit Recombinant Monoclonal NFM antibody. Suitable for ICC/IF, IP, Flow Cyt (Intra), ELISA, WB, IHC-Fr, IHC-P and reacts with Mouse, Rat, Human samples. Cited in 2 publications.

Key facts

Isotype
IgG
Form
Liquid
Clonality
Monoclonal
Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number
EPR23510-76
Purification technique
Affinity purification Protein A
Concentration
Loading...

Storage

Shipped at conditions
Blue Ice
Appropriate short-term storage duration
1-2 weeks
Appropriate short-term storage conditions
+4°C
Appropriate long-term storage conditions
-20°C
Aliquoting information
Upon delivery aliquot
Storage information
Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.
Activity summary

The 160 kD Neurofilament Medium also known as NEFM NF-M or NEF-M belongs to the family of neurofilament proteins. This protein is an important structural component of neurons particularly in the axonal cytoskeleton. Neurofilament Medium has a molecular mass of around 160 kilodaltons (kD). It shows high levels of expression in neuronal tissues contributing to the maintenance of axonal caliber. Commonly research recognizes it alongside other neurofilament proteins emphasizing its role in providing structural integrity to neurons.

Biological function summary

Neurofilament Medium participates in the formation of a stable network of neurofilaments within neurons. Acting as a part of the intermediate filament protein family it forms a complex with other neurofilaments such as NF-L and NF-H. This complex supports neuron structure and plays an important role in axonal transport. The heteropolymeric nature of neurofilaments contributes to their mechanical stability facilitating essential neuronal functions by aligning neurofilaments along the axon.

Pathways

Neurofilament Medium integrates into the cytoskeletal arrangement pathways that govern axonal transport and stability. It plays a significant role in the neuronal transport pathway associating with other proteins such as dynein and kinesin responsible for the motor functions along axons. Also it relates to the MAP kinase pathway where phosphorylation events modulate its assembly and disassembly dynamics in response to cellular signals.

Associated diseases and disorders

Neurofilament Medium closely relates to neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Aberrant phosphorylation or aggregation of NEFM can disrupt neuronal function leading to pathology. In ALS the abnormal accumulation of neurofilaments correlates with motor neuron degeneration. In Alzheimer's its interaction with tau protein poses significant interest as altered states of either could exacerbate neurofibrillary tangles worsening cognitive decline.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

21 product images

  • Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Western blot staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The molecular weight is consistent to what has been described in the literature (PMID:19239416, PMID:27000625).

    Negative control: liver (PMID:30541916).

    Exposure time: 10 seconds.

    All lanes: Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348) at 1/1000 dilution

    Lane 1: Human cerebellum tissue lysate at 20 µg

    Lane 2: Human nerve tissue lysate at 20 µg

    Lane 3: Human liver tissue lysate at 20 µg

    Secondary

    All lanes: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution

    Predicted band size: 102 kDa

    Observed band size: 160 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Positive staining on human cerebellum.

    Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of paraffin-embedded mouse liver tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Negative control: No staining on mouse liver.

    Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunofluorescent analysis of parental HEK-293T (EDWT04) and NEFM KO HEK-293T (ED040746) cells labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 μg/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was (Blue).

    Confocal image showing cytoplasmic staining in parental HEK-293T cells and no staining in NEFM KO HEK-293T cells.

    Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

  • Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 μg with ab254348 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.

    Lane 1: Mouse brain tissue lysate 10 μg.

    Lane 2: ab254348 IP in mouse brain tissue lysate.

    Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab254348 in mouse brain tissue lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 3.25 secs.

    All lanes: Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    Predicted band size: 102 kDa

    Observed band size: 160 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunofluorescent analysis of mouse primary neural/glia cells fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100, labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 μg/ml) dilution. Anti-MAP2 antibody [HM-2] - Neuronal Marker ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 (4 μg/ml). Followed by secondary Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 (2 μg/ml) (Green). Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) was used as secondary counterstain at 1/1000 (2 μg/ml) (Red). The nuclear counterstain was DAPI (Blue).

    Confocal image showing cytoplasmic staining in mouse primary neuron cells.
    Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

    Negative Control 1: Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 1/1000 (2 μg/ml)

    Negative Control 2: Anti-MAP2 antibody [HM-2] - Neuronal Marker ab11267 1/500 (4 μg/ml), secondary: Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 1/1000 (2μg/ml)

  • Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunofluorescent analysis of rat primary neural/glia cells fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100, labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 μg/ml) dilution. Anti-MAP2 antibody [HM-2] - Neuronal Marker ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 (4 μg/ml). Followed by secondary Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 (2 μg/ml) (Green). Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) was used as secondary counterstain at 1/1000 (2 μg/ml) (Red). The nuclear counterstain was DAPI (Blue).

    Confocal image showing cytoplasmic staining in rat primary neuron cells.
    Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

    Negative Control 1: Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 1/1000 (2 μg/ml)

    Negative Control 2: Anti-MAP2 antibody [HM-2] - Neuronal Marker ab11267 1/500 (4 μg/ml), secondary: Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 1/1000 (2 μg/ml)

  • Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Flow Cytometry (Intracellular) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized wild-type HEK-293T (human embryonic kidney epithelial cell, Right)/ 160 kD Neurofilament Medium knockout HEK-293T cells (Left) labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 μg). A Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.

    Positive staining on human wild-type HEK-293T cell line (ab255449), while no staining on human NEFM knockout HEK-293T cells (Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line ab266741).

  • Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Frozen sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

    Positive staining on mouse cerebrum is observed.

    Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488 )at 1/1000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

  • Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Frozen sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (blue).

    Negative control: No staining on mouse liver (PMID: 30541916) is observed.

    Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

  • ELISA - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    ELISA - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    ELISA analysis using ab254348 at a range of 0-1000 ng/ml followed by a Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L）at 1/2500 dilution. Antigen concentration: 1000 ng/ml.

    Antigens: Mouse 160 kD Neurofilament Medium.

  • Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Western blot staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The molecular weight is consistent to what has been described in the literature (PMID:19239416, PMID:27000625).

    Negative control: liver (PMID:30541916).

    Exposure times: Lane 1-3: 3.25 secs; Lane 4-6: 10 secs.

    All lanes: Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348) at 1/1000 dilution

    Lane 1: Mouse brain tissue lysate at 20 µg

    Lane 2: Mouse cerebellum tissue lysate at 20 µg

    Lane 3: Mouse liver tissue lysate at 20 µg

    Lane 4: Rat brain tissue lysate at 20 µg

    Lane 5: Rat cerebellum tissue lysate at 20 µg

    Lane 6: Rat liver tissue lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Predicted band size: 102 kDa

    Observed band size: 160 kDa

  • Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Western blot staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

    Lanes 1-3: Merged signal (red and green). Green - ab254348 observed at 160 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.

    ab254348 Anti-NEFM antibody [EPR23510-76] was shown to specifically react with NEFM in wild-type HEK-293T cells. Loss of signal was observed when the knockout cell line Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell line ab266741 (knockout cell lysate Human NEFM (160 kD Neurofilament Medium) knockout HEK-293T cell lysate ab257103) was used. Wild-type and NEFM knockout samples were subjected to SDS-PAGE. ab254348 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated at at 4°C overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

    All lanes: Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348) at 1/1000 dilution

    Lane 1: Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

    Lane 2: NEFM knockout HEK-293T whole cell lysate at 20 µg

    Lane 3: A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution

    Predicted band size: 102 kDa

    Observed band size: 160 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Positive staining on human cerebrum.

    Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Positive staining on mouse cerebellum.

    Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium was immunoprecipitated from 0.35 mg rat brain tissue lysate 10 μg with ab254348 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.

    Lane 1: Rat brain tissue lysate 10 μg.

    Lane 2: ab254348 IP in rat brain tissue lysate.

    Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab254348 in rat brain tissue lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 5.5 secs.

    All lanes: Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    Predicted band size: 102 kDa

    Observed band size: 160 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Positive staining on rat cerebellum.

    Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Frozen sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

    Positive staining on rat cerebrum is observed.

    Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488 )at 1/1000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

  • Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Immunohistochemistry (Frozen sections) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (blue).

    Negative control: No staining on rat liver (PMID: 30541916) is observed.

    Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

    Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

  • Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Flow Cytometry (Intracellular) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized mouse primary neuron cells labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 μg)/ Right compared with a Rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Left.

    A Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.

  • Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

    160 kD Neurofilament Medium Flow Cytometry (Intracellular) staining using rabbit Anti-160 kD Neurofilament Medium antibody

    Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized rat primary neuron cells cells labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 μg)/ Right compared with a Rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Left.

    A Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.

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