Overview

  • Product name
    Anti-Hypophosphorylated Neurofilament H antibody [N52]
  • Description
    Mouse monoclonal [N52] to Hypophosphorylated Neurofilament H
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IHC-P, IHC-Fr, IHC-FoFr, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Other Immunogen Type corresponding to Pig Hypophosphorylated Neurofilament H (C terminal). C-terminal segment of enzymatically dephosphorylated pig Neurofilament 200.

  • General notes

    Other applications have not been tested.


    Optimal dilutions should be determined by end users.

     

Properties

Applications

Our Abpromise guarantee covers the use of ab82259 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 0.5 µg/ml. Detects a band of approximately 200 kDa (predicted molecular weight: 112 kDa).
IHC-P Use a concentration of 1 - 2 µg/ml.
IHC-Fr Use a concentration of 1 - 2 µg/ml. Acetone fixed.
IHC-FoFr 1/50.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

ICC/IF Use at an assay dependent concentration.

Target

  • Function
    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.
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Post-translational
    modifications
    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.
  • Information by UniProt
  • Database links
  • Alternative names
    • 200 kDa neurofilament protein antibody
    • Nefh antibody
    • Neurofilament heavy polypeptide antibody
    • Neurofilament triplet H protein antibody
    • NF-H antibody
    • NF200 antibody
    • NFH antibody
    • NFH_HUMAN antibody
    see all

Images

  • IHC analysis of Hypophosphorylated Neurofilament H using ab82259. Hypophosphorylated Neurofilament H was detected in paraffin-embedded section of rat brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml ab82259 overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex with DAB as the chromogen.

  • ab82259 staining Hypophosphorylated Neurofilament H in Rat nerve tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, permeablized with 0.2 Triton-X and blocked with 10% serum. The sample was incubated with primary antibody (1/50 in PBS plus 1x Casien) at 25°C for 2 hours. An Alexa Fluor®488-conjugated Goat anti-mouse IgG polyclonal(1/200) was used as the secondary antibody. GFAP was labelled with Texas Red.

    See Abreview

  • ab82259 staining hypophosphorylated Neurofilament H in paraffin-embedded rat brain tissue.
  • All lanes : Anti-Hypophosphorylated Neurofilament H antibody [N52] (ab82259) at 0.5 µg/ml

    Lane 1 : rat brain tissue lysates with 5% Non-fat Milk/ TBS
    Lane 2 : mouse brain tissue lysates with 5% Non-fat Milk/ TBS

    Lysates/proteins at 50 µg per lane.

    Secondary
    All lanes : goat anti-mouse IgG-HRP secondary antibody at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 112 kDa
    Additional bands at: 200 kDa (possible post-translational modification)

  • ICC/IF image of ab82259 stained PC12 cells. The cells were 4% formaldehyde 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 (ab82259, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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.

  • Overlay histogram showing SH-SY5Y cells stained with ab82259 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab82259, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:
  • Prest TA  et al. Nerve-specific, xenogeneic extracellular matrix hydrogel promotes recovery following peripheral nerve injury. J Biomed Mater Res A 106:450-459 (2018). IHC-P ; Rat . Read more (PubMed: 28891122) »
  • Shenoy PA  et al. The Somatostatin Receptor-4 Agonist J-2156 Alleviates Mechanical Hypersensitivity in a Rat Model of Breast Cancer Induced Bone Pain. Front Pharmacol 9:495 (2018). Read more (PubMed: 29867498) »
See all 13 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Penguin Tissue sections (brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: tris-EDTA, pH9.0
Permeabilization
No
Specification
brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted May 02 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Zebra finch Tissue sections (brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: tris-EDTA, pH9.0
Permeabilization
No
Specification
brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Apr 26 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (rat motor neuron primary cells from embryo spiral)
Permeabilization
Yes - 0.2% triton
Specification
rat motor neuron primary cells from embryo spiral
Blocking step
Leica Universal IHC blocking as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Aug 06 2015

Application
Immunohistochemistry (Frozen sections)
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Sample
Rat Tissue sections (spinal cord)
Specification
spinal cord
Permeabilization
No
Fixative
Acetone

Abcam user community

Verified customer

Submitted Mar 24 2015

Answer


The antibodies mentioned above are suitable for Rat as well as human samples. ab9018 is yet to be tested in IHC-Fr however as it is tested in IHC-FoFr so it is more likely to work.

The suitable secondary's are for ab4648, ab82259 and ab9018 are

https://www.abcam.com/rabbit-polyclonal-secondary-antibody-to-mouse-igg-hl-hrp-ab97046.html

https://www.abcam.com/goat-polyclonal-secondary-antibody-to-mouse-igg-hl-hrp-ab97023.html

for ab18258 it is

https://www.abcam.com/goat-polyclonal-secondary-antibody-to-rabbit-igg-hl-hrp-ab6721.html

for ab5392 it is;

https://www.abcam.com/goat-polyclonal-secondary-antibody-to-chicken-igy-h-l-hrp-ab97135.html

The protocols are:

https://www.abcam.com/index.html?pageconfig=resource&rid=11417

https://www.abcam.com/index.html?pageconfig=resource&rid=11383

https://www.abcam.com/index.html?pageconfig=resource&rid=13046

IHC-Fr is immunohistochemistry on frozen section

IHC-FoFr is PFA perfusion fixed frozen sections.

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Question

Dear Supplier,

Please see technical questionnaire attached provided by the customer regarding technical issue with AB82259, Anti-Hypophosphorylated Neurofilament H antibody [NF-200.

This was purchased on our PO# PO-12555, dated 05/12/11.

Customer has been advised that this technical inquiry is well over 6 months Abpromise guarantee. I have also requested for any available images, she will email once she find them.





Antibody Code: AB82259

Batch Number: GR190-9



GENERAL INFORMATION

Antibody Storage Conditions (temperature/reconstitution etc)


-200C

Description of the Problem (high background, low signal, non-specific staining etc)



Low and patchy signal.


Sample (Species/Tissue/Cell Type/Cell Line etc)


Nerve tissue

Fixation of Sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration)



Paraformaldehyde

Antigen Retrieval (Enzymatic method, Heat mediated technique etc)



Enzymatic method




Permeabilization step


10% triton during the blocking step

Blocking Conditions (Buffer/time period, Blocking agent)



Blocked in normal goat serum for 30mins

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)



Abcam (ab82259) NF200. Dilution = 1:200 in normal goat serum. Incubation time- overnight followed by washing in 0.1M PBS.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)



Invitrogen alexa fluor 488 goat anti-mouse secondary antibody. Dilution used = 1:1000 in normal goat serum. Incubation time = 2hours. Followed by washing slides in 0.1M PBS.

Detection Method



Fluorescent labeling

Positive and negative controls used (please specify)



Positive control was used.

OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)




How many times have you tried the IHC?


Several times




Have you run a “No Primary” control? No (Delete one)



Do you obtain the same results every time? No (Delete one)



What steps have you altered?


We have tried using different dilution factors and also with and without the antigen retrieval step. When we used a different primary antibody on the same tissue we obtained a better result.

Additional Notes



Document attached with image.

Thanks


Kind Regards,

Read More
Answer

Thank you for your enquiry regarding ab82259 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) It would useful to get an image (saved as a jpeg file) from the customer.

2) Sample type: Nerve tissue

Questions:

- Is the sample from central or peripheral nerve tissue?

- From which species?

3) Antigen retriaval:Enzymatic method can damage the tissue structure of it is not optimised. Has the customer tried HIER and if so for ho w long?

4) Permeabilization: 10% triton

The concentration is extremely high and it may well be the main factor causing the unexpected staining pattern. For immunostaining Tween 20, Saponin, Digitonin and Leucoperm from 0.2 to 0.5%. Customer can find some useful information at this site:

https://www.abcam.com/index.html?pageconfig=resource&rid=11462

I hope this will be useful for you and for your customer.

Read More

Answer

I am very pleased to hear you would like to test ab5076, ab8049, ab18258, ab58802, ab64581, ab82259 and send us your results. The testing discount doesn't apply to ab4674 since this has already been tested in Rhesus monkey and IHC so we would guarantee that experiment to work.

The codes will give you a value off your next orders before the expiration date. To redeem this offer, please submit 6 Abreview with rhesus monkey data and include the code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview wit has been submitted and can be redeemed in one of the following ways:
1) Call to place your order and mention the code to our customer service department;
2) Include the code in your fax order;
3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated. Remember if the product does not work you will be covered by our Abpromise guarantee and eligible for a full refund or replacement.

If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: https://www.abcam.com/abtrial.

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Answer

Thank you for contacting us.

Yes, the antibody ab82259 can be used to stain sections of paraffin-embedded tissue but the paraffin will need to be removed from the sections. Without paraffin removal, aqueous solutions will not diffuse into the tissue.

This is typically accomplished by moving the slides through a series of solvents, for 3 minutes each: 2 containers of xylene (or a non-toxic alternative such as Slide Brite), and a graded series of ethanol:water mixtures (100% ethanol, 80%, 70%, 50%, distilled water).

At this point, the sections are "re-hydrated" and are ready for immunostaining.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Rat Tissue sections (nerve)
Specification
nerve
Fixative
Formaldehyde
Permeabilization
Yes - 0.2% triton

Abcam user community

Verified customer

Submitted May 08 2013

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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