Anti-CD3 antibody [PS1] (ab699)

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Die Konzentration für den ab699 Anti-CD3 antibody [PS1] mit dem Lot GR95849-1, welches Sie erhalten haben, beträgt 80ug/ml.

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I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement (ab699 - as an exchange of the original ab7507) with the order number 1169598.

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Thank you for your response.

We sell the same clone under ab699 code number. You may wish to take a look at the datasheet to see if it is suitable for your need.

https://www.abcam.com/ab699: Anti-CD3 antibody [PS1]

Let me know how you wish to proceed? I look forward to hearing from you soon.

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TheIgG concentration ofab699 is lot specific, but the total protein concentration is ˜ 10 mg/mL for all lots. I would be happy to provide the specific concentration if you let me know your lot number.
I hope this helps, please let me know if you need any additional information or assistance.

I have now reviewed the literature you have sent me and am a little more familiar with what you are trying to achieve. Currently it looks as if you are getting quite specific staining of the T cells in the tip of the villi of the healthy patient, and similar localisation of the dendritic cells but with higher background in this case. I think what you need to do in order to be sure of the staining is to perform a "no-primary" control. I would do this at the concentration you have been using (1/400) with exactly the same blocking conditions and wash steps applied. If you observe background, as I suspect you may do, certainly in the case of CD11c, I would reduce the concentration of the secondary antibody to 1/1000 and possibly even 1/2000 and see if this non-specificity is reduced. I would also suggest with your future experiments to use 10 % serum from the same species of the secondary antibodies being used if possible. Otherwise increase the BSA blocking buffer from 1% to 3 %. I would also optimise if incubation overnight at 4°C or 1-2 hours at room temperature produces the best results. I hope these suggestions are helping. Let me know how the "no primary" controls turn out and if you have any further questions please do not hesitate to ask. 

Thank you for the very swift reply. From first inspection it looks as if the CD3 staining is much improved and there is specific staining but in order to confirm this I will need to look into it a little further. This would be greatly helped if you could explain a little about what you are trying to achieve with the experiments being performed and if you have any relevant literature I could look over this would also be very helpful. I look forward to your reply.

I am currently looking into your new results. What would help me though is if you could outline briefly what your experiments are trying to achieve and if you have any relavent references I can look over in regards to this. I also have a few further questions. Did you do the "no primary" staining. If so could you share these images with me. Last time I gave you a number of suggestions which may be worth trying, could I just confirm the conditions used for this new expreiment: 1. was a blocking step introduced? If so which conditions did you used? 2. what incubation times and temperatures were used for the primary and secondary? 3. what dilution buffer was used for the primary and secondary antibodies? 4. what concentration of the secondary antibodies was used? Hopefully with this information I can further advise you but on first inspection there does appear to be an improvement.

It would be interesting to see the "no primary" control slides. Having looked at the secondary antibodies that you are using I would maybe also suggest trying a lower dilution of these. The datasheet suggest a 1/1000 dilution for both antibodies, if you are still seeing non-specific staining I would try using the antibodies at this dilution and maybe trying 1/2000 as well. It may be easier for you to work with one of the stainings at a time and once you have seen improvements in one, the optimisation of the other one may be quicker. If you have any further questions please do not hesitate to ask. I look forward to hearing of your progress.

Thank you for contacting us. I'm sorry that the staining currently isn't working as you would like. There are a few suggestions that may help the results. Firstly I came across this description of how to prepare frozen sections. I thought it quite interesting (if a little wordy) but particularly in regards to the importance in quickly preparing samples to achieve good, specific staining. http://www.ihcworld.com/_protocols/histology/frozen_section_technique_1.htm Additionally, as you say it would be worth trying a few different dilutions for the primary antibodies. Although counter intuitively I would suggest trying higher concentrations: 1/50, 1/100, 1/250 as these antibodies are not very highly concentrated. Another thing that may improve the non-specific binding considerably is introducing a separate blocking step. Try incubating the sample with 10% serum from species which secondary antibody was raised in or 3% BSA in PBST for 30 min. I would also see if the incubation time with the antibody has an effect, try both 1 hour at room temperature and as you are doing, overnight at 4°C. I would continue to use 1% BSA in both the primary and secondary antibody diluent as you are doing currently. With washing using PBS between each step. As you have performed a "no primary" control, were the results of this clean? I was a little confused as to what secondary antibodies you are using. Would you be able to supply the catalog numbers (even if from other companies). Before carrying out the double staining I would try to optimise the staining for each antibody then combine the protocols after this has been achieved. I hope this information has been helpful. If you have any further questions please do not hesitate to ask. I look forward to hearing if there are any improvements.