I'm sorry but I didn't use anti-CD28. Have you search in "Biocompare antibody" site?

Dear Shibabrata,

Can you please tell me which secondary antibody are you using? It it fluorescent?

I don't know if there are any CD28-antibodies working on FFPE-sections. I guess you don't have a chance to obtain fresh samples for frozen sections, right?

Since you probably know frozen sections may have other epitopes than FFPE-sections due to fixation artifacts. Antigen retrieval should restore the "natural" epitope but you don't have a guarantee for that.

There are also several protocols (e.g. heat, pH, enzymatic...) for antigen retrieval and not all protocols work for all tissues/epitopes even if a singular protocol works for most. It it mainly trial-and-error to find a combination of fixation+retrieval+antibody that works. If you find a combination, don't forget your positive and negative controls to show that your reaction is specific and not just due to a "retrieval artifact"!

Good luck!

Thank you all for your valued reply.

Dear Paola: I have searched biocmpare and found CD28 purified from BD that I have used.

Dear Alexandre: I am using the Dako envission system where the secondary is an anti-mouse/rabbit LINK polymer.

Dear Vivica: Thank you for your suggestion. We are actually collecting samples for frozen section. Actually we are working on patient skin and PKDL, the disease we are working on, is pretty rare we get 5-6 patients a year. So, the samples we have, we wanted to try on them, but now we are changing.

I have used the heat mediated retrival. However i will try the enzymatic process as you suggested. Thanks again. I will update the result to you.

Dear Shibabrata,

If the problem is not getting any bands then I advise you to use a high sensitivity detection kit (I don't have the reference here but I can get it tomorrow, it does miracles when using difficult antibodies).

The antigen retrieval methods can be tricky depending on the tissues, it may result in extra background. Try with and without antigen retrieval. I usually use a the heat mediated retrieval with a microwave or pressure cooker.

I even think we might be using a CD28 antibody in the lab, I'll figure that out tomorrow as well.

Good luck, Alex

Dear Alex,

Thank you for the answer. I will be highly obliged if you provide the details. I have use the heat mediated antigen retrival too.

Thank you,

Shibabrata

Dear Shibabrata,

I checked and I'm afraid we don't have the CD28 antibody in the lab.

Regarding the kit I have mentioned, it's the SuperSignal® West Femto Maximum Sensitivity Substrate from Pierce Biotechnology. This signal amplification kit works miracles with some antibodies. Maybe it can help you just the same way it did for me.

Good luck

Dear Alex

Thank you very much for the help and reply I will definitely try and inform you.