重组Anti-beta II Tubulin抗体[7B9] (ab28035)

Thank you very much for your email.

Firstly I would like to confirm that the Alexa-488 goat anti-mouse secondary antibodyshould be compatible with anti tubulin antibody ab28035 or ab28036as well. It should also be compatible with work on rat/mouse/human cardiomyocytes. Using an anti mouse antibody on mouse tissue can stain the endogenous immunoglobulins and therefore lead to high background. I am however not sure whether in the freshly isolated cardiocytes there would be any immunoglobulins present.
It might can be useful to use a secondary antibody which has been crossabsorbed against rat immunoglobulins, so it would not recognize the endogenous rat immunogloblins. As per your request, here are some suggestions either excitable at 561nm or 633nm:

-Cy5 conjugated: ab6563 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Cy5 ®), pre-adsorbed against many species, including rat https://www.abcam.com/Goat-Mouse-IgG-HL-Cy5--preadsorbed-ab6563.html (or use the following: https://www.abcam.com/Goat-Mouse-IgG-HL-Cy5--preadsorbed-ab6563.html).
-APC conjugated; ab130782 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Allophycocyanin), pre-adsorbed only against human https://www.abcam.com/Goat-Mouse-IgG-HL-APC-preadsorbed-ab130782.html (or use the following: https://www.abcam.com/Goat-Mouse-IgG-HL-APC-preadsorbed-ab130782.html).
-DyLight 550 conjugated: ab98713 Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (DyLight® 550), pre-adsorbed against human and rat https://www.abcam.com/Goat-Mouse-IgG-Fc-DyLight-550-preadsorbed-ab98713.html (or use the following: https://www.abcam.com/Goat-Mouse-IgG-Fc-DyLight-550-preadsorbed-ab98713.html).
-Texas Red conjugated: ab7066 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (TR), pre-adsorbed against many species, including rat https://www.abcam.com/Goat-Mouse-IgG-HL-Texas-Red-preadsorbed-ab7066.html (or use the following: https://www.abcam.com/Goat-Mouse-IgG-HL-Texas-Red-preadsorbed-ab7066.html).
-Texas Red conjugated F(ab')2 fragment: ab5884 Goat F(ab')2 polyclonal Secondary Antibody to Mouse IgG - (Fab)'2 (TR), pre-adsorbed against many species, also against rat https://www.abcam.com/Goat-Fab2-Mouse-IgG-Fab2-Texas-Red-preadsorbed-ab5884.html (or use the following: https://www.abcam.com/Goat-Fab2-Mouse-IgG-Fab2-Texas-Red-preadsorbed-ab5884.html).

Theuse ofF(ab')2 fragment will further reduce the background, if there is some background problem due to unspecificFcportion binding of the secondary antibody. I can therefore recommendthe ab5884.

I have also through the protocol and would like to raise the following points in order to understand the procedure better and toprovide recommendationsin regards to the primaryantibody usedwhich might can help to improve the results:

1.) Can you please confirm which antibody you are using, the ab28035 or the ab28036?

2.) For both antibodies, the fixation method for the ICC has been methanol. I would therefore strongly recommend to use also methanol for fixation if this is possible for you. Indeed, PFA and methanol do fix by different mechanism. The PFA fixes by crosslinking covalently the proteins whereas the methanol fixes by precipitating the proteins. The two mechanism can therefore expose different epitopes and work differently for different antibodies. As we have only tested methanol so far, I can recommend to test also methanol.

3.) I can also recommend to reduce the fixation time for PFA to 15 minutes for primary cell cultures, as overfixation can also reduce the staining.

4.) I can also recommend to test different blocking substances, such as gelatin and serum (of the species in which the secondary antibody has been raised) as alternatives to the BSA to see whether this reduces the background.

5.) In order to reduce the background, I can also recommend to use PBS with 0.05% Tween in the washing buffer. This will help to wash the slides.

I hope this information are helpful. Please do let me know your thoughts and comments. I am looking forward to hear back from you.

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products.

As a positive control I can recommend the HeLa cells. Indeed, this antibody has been tested on humantissue so farand on the datasheet you will find a HeLa cell image. https://www.abcam.com/beta-II-Tubulin-antibody-7B9-ab28035.html (or use the following: https://www.abcam.com/beta-II-Tubulin-antibody-7B9-ab28035.html). The antibody has not yet been tested in immunohistochemistry nor on mouse tissue.We do therefore not know whether this antibody can workon this samples, it might well be though.

I would be pleased tohelp you to troubleshoot and optimise the protocol ifyou wish. I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results.

I look forward to receiving your reply. Please do let us know also if you have any other question.