Anti-Oligodendrocyte Specific蛋白抗体- Oligodendrocyte Marker (ab7474)

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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.
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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.

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.

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.

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.

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.

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.