Anti-Oligodendrocyte Specific Protein antibody - Oligodendrocyte Marker (ab7474)

Overview

  • Product name
    Anti-Oligodendrocyte Specific Protein antibody - Oligodendrocyte Marker
    See all Oligodendrocyte Specific Protein primary antibodies
  • Description
    Rabbit polyclonal to Oligodendrocyte Specific Protein - Oligodendrocyte Marker
  • Host species
    Rabbit
  • Specificity
    Reacts specifically with 22 kDa protein derived from CNS samples.
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, WB, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Mouse, Rat
    Predicted to work with: Human
  • Immunogen

    A 15 residue synthetic C-terminal peptide.

  • Positive control
    • WB: rat CNS and mouse brain and testis
  • General notes
    The concentration depends on the batch, please enquire for current stock concentration.

Properties

Applications

Our Abpromise guarantee covers the use of ab7474 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
IHC-P 1/50 - 1/500.
IHC-Fr Use at an assay dependent concentration.
WB 1/200 - 1/5000. Detects a band of approximately 22 kDa (predicted molecular weight: 22 kDa). Block in Tris-buffered saline (TBS) containing 1% BSA overnight at 4C. Possible oligomerisation or polimerisation of the protein have been reported and can lead to high MW bands.
IHC-FoFr 1/100.

Target

Images

  • Immunofluorescent staining for OSP obtained with OSP antibody (ab7474) in rat brain cortex. The picture shows oligodendrocyte immunostaining in the cingular cortex. Picture taken with X20 objective, cells stained are ~30microns in diameter. Animals were intracardially perfused with 4% PFA. Tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. Protocol : free floating IHC on 30um cryostat sections. Primary antibody ab7474 was used at 1/100 and incubated overnight at RT. Secondary antibody, Alexa fluor 488 at 1/1000, incubated for 2h at RT.
  • IHC image of ab7474 staining in human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7474, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Immunohistochemical analysis of adult mouse testis tissue, staining Oligodendrocyte Specific Protein (red) with ab7474.

    Tissue was blocked with 5% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/100 in 1% BSA) for 1 hour at 25°C. A TRITC-conjugated donkey anti-rabbit polyclonal IgG (1/200) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Zhao X  et al. Neutrophil polarization by IL-27 as a therapeutic target for intracerebral hemorrhage. Nat Commun 8:602 (2017). Read more (PubMed: 28928459) »
  • Matsunaga E  et al. Identification of tool use acquisition-associated genes in the primate neocortex. Dev Growth Differ 57:484-95 (2015). Read more (PubMed: 26173833) »
See all 9 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 0.01M citric acide/sodium citrate
Sample
Sheep Tissue sections (1-2 month old testis)
Specification
1-2 month old testis
Permeabilization
No
Fixative
4%PFA

Abcam user community

Verified customer

Submitted Jan 19 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer pH 6.0
Sample
Human Tissue sections (spinalcord)
Specification
spinalcord
Permeabilization
No
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 27 2014

Answer

Thank you for contacting us.
Oligodendrocyte-specific protein (OSP) is a putative four-transmembrane protein of 22kD. It is primarily expressed in oligodendrocytes of the CNS and Sertoli cells of testes in the adult mouse. OSP is the third most abundant CNS myelin protein and contributes to 7% of the total myelin protein. OSP shares sequence homology with the claudin family of tight junction (TJ) proteins. It forms TJs in cell culture and co-localizes with structures similar to TJs in myelin. Thus, it was renamed OSP/Claudin-11. OSP/Claudin-11-null mice lack intramembranous junctions in between Sertoli cells in testes and within CNS myelin sheaths, producing reproductive and neurological deficiencies. This finding indicates that OSP/Claudin-11 is necessary for the formation of TJs. OSP/Claudin-11 expression is highly regulated during development, suggesting that it may play an important role in growth and differentiation of oligodendrocytes and other cells outside the CNS. OSP/Claudin-11 forms a complex with a novel member of the tetraspanin superfamily (OAP1) and b1 integrin. This egulates proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair. Claudin-11 is also expressed in the epithelial tight junctions of the choroids plexus. OSP/Claudin-11 has been implicated as an autoantigen in the development of autoimmune demyelinating disease.
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 (Frozen sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Sample
Mouse Tissue sections (adult testes)
Specification
adult testes
Permeabilization
No
Fixative
4 %

Dr. Qing Wen

Verified customer

Submitted Dec 24 2012

Question

The Invitrogen secondary antibodies were:

A21449, Alexa Fluor 647, goat anti-chicken:used with your MAP-2 targeting ab (ab5392)

A21448, Alexa Fluor 647, donkey anti-sheep: used with your GFAP targeting ab (ab90601) (never used together with the previous ab in a same staining, obviously)

A11008, Alexa Fluor 488, goat anti-rabbit:used with your OSP targeting ab (ab7474)

A10036, Alex Fluor 546, donkey anti-mouse:used to target or protein of interest in CNS cells

Hope this time you have everything you need.

Please find attached an example of staining obtained with your primary antibodies. It is a .tif format. The staining has been performed on a brain section of human biopsy.

I just send you one example because all the staining looks pretty similar with your antibodies.

In red: a working antibody used to target our protein of interest, revealed with an Invitrogen antibody (A10036) (good staining)

In green: the OSP staining (bad staining, often co-staining in nuclei only, smearing)

In yellow: the GFAP staining (absent, even though astrocytes are for sure present in the section stained)

The yellow staining is absent. If we modify the settings to increase the signal, the yellow staining becomes to appear also in the AbII (secondary antibody only) staining. This true for all staining to have optimal settings and avoid false positive signals. In general, MAP-2 was a little bit better than GFAP but not satisfactory for us either (not shown in the pictures forwarded to you). The data are brut from the confocal, I didn't modify them afterwards to emphasize the staining quality.

Sincerly,

Read More
Answer

Thank you for your message and for providing this further information and the images.

Reviewing the details, I appreciate the time you have spent on these experiments and would be pleased to arrange afree of charge replacementor credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

Read More

Question

Ok I understand your inquiries, but please, I hope there will be no more new people I will have to discuss with.

I will answer step by step to each of your concerns:

1. Yes indeed, it has been ordered through Lucerna Chem company. I just called them and the ordering number they gave me was the B120505 (is it possible, as you gave me the no B120502 which is really close...?). The date of order was indeed the 8th of June.

2. Indeed, we could rule out a secondary antibody problem since they work properly with other antibodies and, furthermore when they are used alone without primary antibody (neg control), no staining can be detected with the proper settings for our samples.

We have been using Invitrogen secondary antibodies, Alexa Fluor (647, 488 and 546) chromophore-linked, which is the most effective and suitable for immunofluorescence.

3. Each time that we switched to a new tissue/cell type, we tested all antibodies on it. We use only human cells. I tested the antibodies on CNS tumor cell lines, then on human brain tissue sections and finally on human brain primo-cultures.

To note: of course, when we coupled several primary and then secondary antibodies in a staining, we carefully avoided any cross-reactivity among the antibodies (primary-primary, secondary-secondary and primary-secondary) regarding the hosts.

4. I can definitely provide pictures, but for this I will have to send another e-mail to you tomorrow, since it will take a bit of time to select the pictures and organize them in a power-point folder, format readable for you.

In the meantime, feel free to ask further questions if my reply did not give all answers to your inquiries.

All the best,

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Answer

Thank you for your reply.

I look forward to receiving the images, I appreciate your cooperation.

I would appreciate if you are also able to provide further information regarding the secondary antibodies, as previously requested. The most helpful would be if you are able to provide theproduct codes or catalog numbers, and details of which primary they were used with.

Many thanks for your time. I look forward to hearing fromyou.

Read More

Answer

Thank you for your reply and for kindly providing this further information.

Reviewing the details, I fully understand your concerns and itregrettable the results fromthese three antibodies have not been satisfactory. It is unusual for 3 antibodies to be giving the same non specific results.Therefore, before proceeding in this case, I would appreciate if you are able to provide some further information which will help my final investigations of this case so we can bring this to a close. This will also be vital for our quality monitoring.

1. Please confirm if you have ordered through Lucerna Chem? In this case, they will be able to provide you with thePO number they used when placing the order with us. I would appreciate if you could contact them and ask them to confirm the order number and date of purchase. Was this B120502 ordered 8th June?

2. Could you confirm if the current vial of secondary antibodies are working well with other primary antibodies? What are the results of the no primary controls for the secondaries? Which secondary antibodies have you been using?

3. Have you been using the same type of samples (same species and tissue) for all three antibodies? If not, please provide further details.

4. I would appreciate if you are able to provide images which will help me to assess the results.

Thank you for your continued cooperation. I look forward to hearing from you with the requested details and hope we can resolve this case as soon as possible.

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Answer

Thank you for your patience. My colleague is still out of office this week.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

In order to proceed with your complaint I would be very grateful to know the following information.

- Could you please confirm your order details (date of purchase, Abcam Order Number or your PON)

- Please also confirm if you are having problem with only ab7474 or other products?

I look forward to hearing from you soon.

Read More

Answer

Thank you for your email.

Fixing in paraformaldehyde for more than 10-15 min will cross link the proteins to the point where antigen retrieval may be required to ensure the antibody has free access to bind and detect the protein. There is also possibility of non-specific binding of antibody and false results.

It is indeed possible that problem might be due to antibodies however we can never be sure without doing experimental optimizations; also when multiple antibodies fail, the possibility of problem with the protocol or sample is more than antibodies. I would strongly suggest doing a quick experiment with just 10-15 minutes of PFA fixation. If the results do not improve then replacement antibody can be tried.

GFAP is a best astrocyte marker however GLT-1, GLAST, S100 or SC1 can also be tried.

Oligodendrocyte markers could be ab Olig2, CNPAse, Myelin-Oligodendrocyte Glycoprotein (MOG), Carbonic Anhydrase II or Microtubule-Associated Protein 4 etc.

Doublecortin, Tyrosine Hydroxylase, BLBP, Calbindin, PGP9.5, GAD67 or GAP43 are other neuronal markers.

PBS plus saponin is fine for antibodies dilution or permeabilization.

I hope these suggestions would be helpful. Should the results do not improve please do not hesitate to contact me for further assistance.

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Question
Answer

Thank you for your email and I am sorry to hear about the problem you are experiencing.

The protocol seems to fine to me, which is pretty much straight forward so the problem might be with the negative control cells you have chosen; these might not be a proper negative control for Claudin 11. There are publications that actually explains or mention the claudin 11 expression in PBMCs. Please check the links below;

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2169001/

http://jvi.asm.org/content/early/2007/09/05/JVI.01457-07.full.pdf

http://jvi.asm.org/content/early/2007/09/05/JVI.01457-07.full.pdf

The gene expression profile of PBMCs (NK cells) shows the CLDN11 gene is expressed by PBMC. www.nature.com/ni/journal/v12/n8/extref/ni.2067-S5.xlsx

The GFAP antibody ab90601 is well characterized and is guaranteed for ICC/IF. I would suggest doingfollowing protocol optimizations for better results
- 10% Normal serum or 3%BSA with 1-2 hours incubation for blocking
- Higher dilution of primary or secondary antibody
- Fixations of cells with 4% PFA for 10-15 minutes
- Washing cells in PBST for5 minutes wash X 3 times.

I hope this information will be helpful. Should you have any question please do not hesitate to contact me.

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1-10 of 21 Abreviews or Q&A

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