Anti-CD11b antibody [M1/70] (ab8878)

Product code: 8878Inquiry: Our customer wishes to test ab8878 in IHC-FrFl with mouse samples. Are you interested in this type of the information for the Abreview promotion (The customer buys certain antibodies and upload Abreview on Abcam's website and then we give 100% credit note for this antibody to buy next antibody)? If your answer is positive here is customer's

ANSWER:

Thank you for contacting us.
We do not have abreview for IHC-FrFl application. We however consider this application under IHC-FoFr which PFA perfused fixed frozen samples. Could you please request the protocol customer would be using, we can then decide the application?
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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on the vial was written mouse monoclonal - although it is rat antibody
customer used this antibody before and had good staining. same reactives and secondary. mouse brain tumor slides used at 1/200 the 1.3,g/ml antibody
ordered three vials:
two written on it rat monoclonal antibody x
one written on it mouse monoclonal antibody x

ANSWER:

Thank you very much for your call and your patience while we were investigating this issue.

We have checked all our remaining vials and the possibilities of mix up. However we could not find any further errors nor the reason of the initial error. I am very sorry.

Have you had the opportunity to test the other two vials with the correct labelling? Have they provided a satisfactory result? If yes, I will be happy just to replace the third vial. If the two other vials however have not been providing good results, we could maybe check the protocol firstly together to see whether we could find another explanation.

Once again, thank you for your collaboration. I am looking forward to hear back from you.

Phone call requesting suggestions of antibodies which could be used to separate populations of mouse hippocampal glia and neurone cells for subsequent extraction of RNA. Cell surface markers (does not want to perform permiabilisation).

ANSWER:

Sorry for the delay in getting back to you. I have now had a more thorough assessment of the antibodies I suggested to you, in particular how likely they are to work without permiabilisation of yoursamples. This is a summary of the information I have now:

Neuron markers:

NrCAM

The anti-NrCAM antibody (ab24344) which I suggested to yourecognizes an extracellular region of the protein and should therefore me more amenable to detection without permiabilisation, however, as discussed previously this antibody has not been used in flow cytometry/FACS to our knowledge. This does not mean that it will not work, simply that it has not yet been tried. We have no alternative antibody to this targetwhich has been used in flow cytometry but I would be happy to offer you a testing discount if you are interested in trying ab24344 for your experiments.

NSE

When I looked back on my suggestions of anti-NSE antibody (ab53025) I discovered that this antibody, whilst it had been used with flow cytometry from the submission of an Abreview, the cells used had been fixed in paraformaldehyde and permiabilisted with methanol. Looking further into it, all other reports of its use had also used a permiabilisation agent, I therefore cannot guarantee that it would work without. The immunogen used to raise this antibody was derived from human NSE however, from SwissProt the regions of the domains remains undefined and I am therefore unsure if the region the antibody detects is from the extracellular domain of the protein.

Looking through our other anti-NSE antibodies, ab16808, may be a better option as although again Ido not know for certain which region is bound, the antibody has has been used with flow cytometry with 4% paraffin fixation, without permiabilisation.

GAD67

The anti-GAD67 antibody I suggested (ab26116) has been used forboth flow cytometry and ICC withmethanol fixation used and for the latter technique permiabilisation was also applied. The immunogen usedto raise this antibody is a synthetic peptide corresponding to amino acids 87-106 of Human GAD67 but again the regions have not been very well defined and I cannot therefore be sure that the antibody would be likely to work without permiabilisation.


Afurther targetto consider wouldbe L1CAM. The antibody ab24345 has been used todetect the proteininPC12 cells with4% paraformaldehydefixation,without the need forpermiabilisation. This may also thereforebe a suitablecandidate.


Glia markers

CD45

On checking again the anti-CD45 antibody I suggested (ab10558), whilst flow cytometry had been performed with KM-H2 cells without fixation or permiabilisation, theimmunogen used is taken from the putative cytoplasmic domain (residues 900-1000).I have also found a further antibody against CD45 which may be worth considering,ab10559. This antibody has been used again without fixation or permiabilisation to perform flow cytometry with human cell line KM-H2.

CD11b

The antibody I suggested against this target, ab8878, has been used in flow cytometry without permiabilisation as well as with IHC with paraformaldehyde fixation without permiabilisation. I am seeking further information on exactly how the flow cytometry experiment was carried out.

I'm sorry if this is a bit too much information to digest but I want you to be able to make an informed choice on the antibodies you decide to purchase. With all of the antibodies that I have discussed, particularly the antibodies only previously used withfixation, cannot be guaranteed that they will work without. It may be that the antibody preferentially detects the denatured protein.

I’m sure you have already been looking into how to perform the RNA isolation following the FACS, however in looking into your query I came across this link which may be of use to you:

http://www.immunology.utoronto.ca/FlowCytometry/rna_isolation.htm

As well as this paper which shows the useof paraformaldehyde asa fixativeprior toFACS and mRNAisolation:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017536

As I receive more information on how the flow cytometry experiments were carried out for ab16808 and ab8878 I will let you know. If you would like to participate in the testing discount scheme please let me know and I will arrange a discount code to be generated.

I hope some of this information has been of help. If you require any further information please do not hesitate to contact me again.

I would like to test these antibodies in rabbit in IHC-P, which has not yet been tested.

ANSWER:

I am very pleased to hear you would like to accept our offer and test ab6678 in rabbit, ab8878 in IHC-P, and ab11862 in rabbit. These codes will give you 1 free primary antibody per code before the expiration date. To redeem this offer, please submit an Abreview for each antibody and include the code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The codes will be active once the Abreviews have 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, whether positive or negative. 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: www.abcam.com/collaborationdiscount.

Sorry, I forgot to give you the reference of the product. This is the ab8878. Kind regards,

ANSWER:

Thank you for contacting us. Thanks for confirming the abID. The antibody was tested in IHC-FR, IP, Flow Cyt and western blot so this is guaranteed to work in these applications. Though the human IHC images are not on the datasheetbut theantibody is fully guaranteed to be used with Mouse, Rabbit or Human samples. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.