Anti-MCP1 antibody [2H5] (ab21396)

Below is a link to a paper that uses clone 2H5 (same clone as ab21396) for acetone fixed, frozen tissue staining (Figure 8).

http://ajprenal.physiology.org/content/280/2/F343

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As shown on the following SwissProtlink, http://www.uniprot.org/uniprot/P14844, the calculated molecular weight of the Rat MCP1 is 16.4 kDa, not 11 kDa as for Human. The differences in the molecular weight observed with WB can be due to post transcriptional modifications such as glycosylation.

I contacted the source of the antibody whoinformed me thatthey have not validated the product in house for western blot. The only data they have for the use of this clone [2H5] in Western Blot come from a publication by Luo, Y. et al., 1999, J. Immunol. 163:3985.

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

Vielen Dank für Ihre Anrufe.
Wie besprochen können wir den MCP1 Antikörper für Western blot und Durchflußzytometrie garantieren (www.abcam.com/Abpromise). In der Immuncytochemie/Immunfluoreszenz (ICC/IF) wurde er noch nicht getestet, aber es bestehen gute Chancen, dass er funktioniert, da er ja für die Immunhistochemie getestet ist.
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Thank you for kindly confirming these details as this enables us to closely monitor the quality of our products. I am glad to learn that the customer expects expression of the protein in these tissues, because following good scientific practise a positive control is paramount which is why reviewers certainly will request it. As the protocol seems absolutely fine to me, I would be pleased to arrange a replacement product free of charge with a new vial of ab21396. I am forwarding these details on to our Distributor’s Team who would be happy to organize this. Thank you for your help and cooperation. Please do not hesitate to contact us if you need anything further.

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this Anti-MCP1 antibody. We have received no complaints about this product therefore I am concerned it is not working for them. The protocol indeed looks absolutely fine to me. However, to fully understand the procedure I would like to ask which material has been used. The google translator gave the translation: Human umbilical cord endothelial cells and smooth muscle cells in rats. Can you confirm this please? It would be especially important to know if it is umbilical cord or spinal cord? Also, I would appreciate if the customer could confirm that MCP-1 is indeed expressed by these tissues. To my knowledge, spinal cord tissues or mixed cortical tissues (as investigated by users of our other MCP-1 antibodies, see respective Abreviews ab25124) might be an appropriate positive control, as would be smooth muscle treated with 100µg/ml glycated LDL (ab9669). If the customer cannot get hold of these controls, I might be able to send a recombinant MCP-1 protein as positive control, such as ab9780 or ab73866. Click here (or use the following: https://www.abcam.com/index.html?datasheet=9780). Click here (or use the following: https://www.abcam.com/index.html?datasheet=73866). As the antibody was purchased in September, the customer is covered by our Abpromise guarantee. Therefore if the antibody does not show any results with the positive control, I would be happy to offer a refund or replacement antibody vial. Please do not hesitate to contact me should you have additional questions.

Thank you for your enquiry. I have checked with our laboratory and it seems that this product has not been tested in paraffin embedded sections. The application that we have stated on the datasheet refers to frozen sections, and tissue/cells grown on slides. However, this does not mean that the antibody will not work in this IHC-P application, just that we have not tested it and therefore, can not guarantee that you will get good results. If you decide to go ahead and purchase this product, we encourage you to submit an Abreview via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers. We reward each Abreview with Abpoints which can be used for discounts off future purchases and gifts. To find out more about our Abreview system, please see the following URL: https://www.abcam.com/abreviews I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry. The following paper has a brief description of a procedure for staining frozen sections of rat retina with MCP-1 antibody. It is not the same antibody as our hamster antibody but the procedure is standard. The important thing is to use a buffer with 0.1% saponin for the whole procedure, because it permeabilizes membranes and allows the antibody access to the MCP-1. http://www.iovs.org/cgi/content/full/42/7/1547 For a secondary antibody, if you want use a horseradish peroxidase detection, use ab6892, goat anti-hamster IgG: https://www.abcam.com/index.html?datasheet=6892 Your sections will need to be blocked against non-specific binding of the anti-MCP-1 antibody to the tissue by first incubating the sections with normal goat serum. Our standard protocol for immunostaining frozen tissue is found at a link on this page: https://www.abcam.com/index.html?section=ihc&pageconfig=technical&intAbID=21396&mode=problems If you want to use a fluorochrome-tagged secondary antibody (eg FITC) e-mail me back and I will recommend one of our anti-hamster conjugates. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your reply. A MCP-1 positive control in tissue is a bit tricky as production usually tracks with disease states (for example trauma or autoimmune disease). One of the best positive controls for MCP-1 in tissue is the central nervous system of rats or mice with experimental autoimmune encephalomyelitis (see Journal of Immunology 156:3017-3023). Other labs used ischemic muscle (see 1996 or J. Surg. Res. 134:145, 2006). If you don't have access to tissues from these disease states, you might consider an adherent cell line such as the small cell lung carcinoma cell line NCI-H69 that has been treated with a drug such as doxorubicin (see Int. J. Cancer 103:380, 2003). You can grow these cells on slides as use as positive controls for IHC pretty easily. I hope that this information helps? If you have any additional questions then please get back in contact with me.