Goat F(ab) Anti-Mouse IgG H&L (ab6668)

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As requested I have included with this email our Mouse on Mouse staining tips. For blocking of endogenous IgG we recommend using Goat F (ab) polyclonal Secondary Antibody to Mouse IgG - H&L (ab6668). More information about this product may be found at:

https://www.abcam.com/goat-f-ab-polyclonal-secondary-antibody-to-mouse-igg-h-l-ab6668.html

I’d like to bring your attention to an offer we are running throughout November. If you order any primary antibody you can receive a RabMAb free, whilst stocks last. Simply quote “RABMAB-XBSMG” in your next primary antibody order. More information on this offer can be found from the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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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The forms which you have requested have been created and are now available on the webpages for each product. For your convenience I have also include these with this email.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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I have spoken with the group in charge of creating product SDS forms on your behalf. They have informed me that they have created this form and it will be posted on the public webpage for the product soon.

You may find this form by going to https://www.abcam.com/goat-f-ab-polyclonal-secondary-antibody-to-mouse-igg-h-l-ab6668.html and clicking the 'SDS' button on the right hand side. I have also included a pdf file of this form with this email.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for contacting us, I apologize for the delay in my reply. I would recommend ab114130 for your purposes. The immunogen for this antibody shows no significant similarity to PDGFA, and it is slightly less likely to react with mouse than our other rabbit polyclonal antibodies. That being said, the mouse protein is fairly homologous with human and there is still a chance that ab114130 may show some reactivity to mouse PGDF BB.
If you do want to use the mouse monoclonal antibody ab51869 on mouse tissue, you may need to perform an additional blocking step to prevent mouse-on-mouse background staining. If mouse-on-mouse background is a problem in your assay, it will be visible when running your no primary antibody or isotype control. To reduce this background, you can perform an additional blocking step after your normal serum block and before the primary antibody incubation. The sections should be incubated for 1 hour with a F(ab) fragment unconjugated secondary antibody to mouse IgG, such as ab6668, at a ˜1:100 dilution at room temperature.
I hope this helps, please let me know if you need any additional information or assistance.

Thank you for your reply.

The pretreatment can be performed using a 1:100 dilution of ab6668 for 1 hour just after the serum block. This should be sufficient for blocking endogenous mouse IgGs in your tissue sections. Please let me know if you need any additional information or assistance.

Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

In addition, we have a Polymer IHC Kit which tackles exactly the problem of a high background which arises from this kind of staining (mouse-on-mouse staining):

https://www.abcam.com/Mouse-on-Mouse-Polymer-IHC-Kit-ab127055.html (or use the following: https://www.abcam.com/Mouse-on-Mouse-Polymer-IHC-Kit-ab127055.html).

ab127055 allows the use of mouse antibodies on mouse tissue to obtain excellent signal intensities and clean background.

Another alternative i could recommend would be using three antibodies in total: Use a Fab-fragment anti-mouse antibody against the H+L chain to block the endogenous mouse IgGs, than incubate with your primaries and (as you suggested) use an isotype specific secondary antibody.

A Fab fragment antibody has the advantage that it can "cover" the endogenous mouse IgG tighter than a whole IgG antibody.

https://www.abcam.com/Goat-Fab-Mouse-IgG-HL-ab6668.html (or use the following: https://www.abcam.com/Goat-Fab-Mouse-IgG-HL-ab6668.html).



I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Thank you for your calls today, and it was a pleasure speaking to you. I apologize for the delay in getting this email out to you.

As we discussed,I would recommend using a secondary antibody that lacks the Fc portion (also called "constant fragment") so the Fc receptors in the tissue can not bind to the secondary antibody. We have the goat anti-mouse F(ab)2 secondary antibody ab102223 which is conjugated to DyLight550, a fluorophoresimilar to the AlexaFluor you're currently using-

https://www.abcam.com/Goat-F-ab-2-polyclonal-Secondary-Antibody-to-Rat-IgG-Fab-2-DyLight-550-pre-adsorbed-ab102223.html

I wouldn't expect a secondary antibody pre-adsorbed against ratto cross-react with endogenous rat IgG, however it could be possible for this to be happening.The anti-mouse F(ab) reagent ab6668 would help reduce this, but you might want to try eliminating other factors first. We didn't discuss this over the phone, but have you already tried diluting the antibody further and increasing washing steps? TBST washes can also help reduce background (versus PBST).

We also discussed the F(ab)2, F(ab), and Fc portions of secondary antibodies, and we have a couple good resources that discuss this further-

https://www.abcam.com/index.html?pageconfig=resource&rid=12825

https://www.abcam.com/index.html?pageconfig=resource&rid=12722

Basically, an antibody looks like a "Y". The Fc portion is the long straight stem of the "Y". Immune cells (macrophages, B cells, plasma cells) naturally bind to this Fc portion, and antibody binding to Fc receptors can cause background. By removing the Fc portion, the remaining antibody is the "V" portion, also called the F(ab)2. When these two sections are separated, they are denoted as F(ab). The F(ab) and F(ab)2 will not bind to the Fc receptors in the tissue.

It might be helpful if you could send some images of the background you're seeing? I could better assess what is contributing to the background and be able to recommend a more specific solution.

I hope this information will be useful, but please let me know if you have further questions and I'll be happy to help. I look forward to hearing from you.

Thank you for contacting us.

Indeed, a mouse-on mouse staining can be quite tricky; however, it is not impossible. The background will depend on the fixation method ( perfusion fixed tissue vs. normally fixed tissue), on the tissue itself ( eg. kidneys vs. bone) and the experimental settings.


One way to make sure to decrease or to completelyerase the background is to block the endogenous mouse IgG with a non- conjugated secondary antibody before you incubate with your target antibody. I would suggest to use a Fab or F(ab)2 fragment antibody in this case as they will bound in a higher density than whole IgG antibodies.

You can find the aforementioned antibodies here:


https://www.abcam.com/Goat-Fab2-Mouse-IgG-Fab2-pre-adsorbed-ab98754.html (or use the following: https://www.abcam.com/Goat-Fab2-Mouse-IgG-Fab2-pre-adsorbed-ab98754.html).

https://www.abcam.com/Goat-Fab-Mouse-IgG-HL-ab6668.html (or use the following: https://www.abcam.com/Goat-Fab-Mouse-IgG-HL-ab6668.html).



Another way is to use our hassle- free Mouse on Mouse (M.O.M) Polymer IHC Kit, which contains a special rodent block solution:

https://www.abcam.com/Mouse-on-Mouse-Polymer-IHC-Kit-ab127055.html (or use the following: https://www.abcam.com/Mouse-on-Mouse-Polymer-IHC-Kit-ab127055.html).


In addition, we also received an Abreview (17 July 2009) from one of our customers who rated this antibody highly in IHC. You can contact the author via our website if you like.



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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Thanks for keeping me updated abouttheprogress. I'm so glad to hear that ab7778 gave positive results!

Please let me know if there is anything else that you might need from me in the future. Have a wonderful weekend!

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I am sorry for the trouble with these antibodies. Please let me know if you have any questions or if there is anything else that we can do for you. Have a great day!