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1. The secondary antibodiesn that I used are Goat anti rabbit dy light 488, procured from xxxxx and the other was goat anti mouse Cy3, procured from xxxxxx.
2. These antibodies have been successfully used in our lab for fluorescent staining.
3. Am still performing validation of primary antibodies procured from you (Abcam: Ab32063 & Ab16813). All the experiments have been done on uterus tissue which is positive for both Er alpha and ER beta expression.
4. Microwave heating with sodium citrate buffer at pH 6.0 was used for the antigen retrieval.
5. Blocking was performed by adding 3% Fetal calf serum and 5% Normal goat serum in PBS with 0.1 triton and incubated for 60 min at room temperature.
6. Use of tritonated PBS itself was considered as a methed for permeabilization.
7. At each sted slides were washed 3 times with PBS.
If you any further question please do not hesitate to contact me by mail or by phone.
Asked on Oct 17 2012
Thank you for your detailed reply. That has been very helpful.
I have a few suggestions to make which may improve the results currently observed, or at least allow us to understand what may be contributing to the problems.
I would firstly try to reduce the non-specific background you have been seeing with the secondary antibodies. This would involve trying the staining again with just the secondary antibodies but optimising the conditions to reduce the non-specific binding you have been seeing. This could involve reducing the concentration of secondary antibody used and the blocking solutions used. I would initially just try with the goat serum at 10% for blocking (as mixing blocking types can lead to increased background) with 0.1% Tween 20. I would also introduce 1% goat serum to the antibody diluents (as well as 0.1% Tween 20). You may also want to consider using a different blocking agent altogether, such as 3% BSA.
If this does not improve the results, you may also want to look into optimising the antigen retrieval used. Too much antigen retrieval can lead to higher background staining.
Another thing to consider is that the ab16813 is of the isoform IgM and I do not think the antibody you are currently using is able to detect this isotype. I would therefore suggest using a secondary antibody specific for mouse IgM.
Once the background has been reduced it may be worthwhile to introduce a separate permiabilisation step when using the primary antibodies to ensure good penetration of the two antibodies into the tissue. You could use 0.2% Triton in PBS for 10 minutes.
I hope this information has been of some help. I would be very interested to hear how you get on.
Until then, I wish you all the best with your research.
Answered on Oct 17 2012