I use rat ant-mouse CD8a Ab(from eBioscience) to stain mouse pancreas and the Ab always stains the cells of secretory acini which looks like "specific binding", the "specific binding" not in islet. The CD8 antibody works well for mouse spleen,thymus, lymph node. I think it's pancreas tissue problem, no matter how I try , like bake slides, deparaffin, antigen retrieval, block, Ab incubation time and temperature, wash….the secretory acini staining always there, some area strong, some weak, no particular pattern(I'm sure it's not CD8 antigen). How can I get rid of the "specific" non specific binding? Thanks.
Dear Qian,
That is a common problem with acinar structures of pancreas and salivary glands which makes immunostaining so difficult. I have worked on both and what helped me was using a prolonged incubation in serum-free protein block (I have used Dako) and background-diminishing antigen diluent from the same source. I am sure that there are similar preparations from other manufacturers, but for pancreas I have only used those two. You may have to do quite a few experiments to establish the working protocol for timing of incubations with protein block and concentration of your prime antibody (after blocking and using this diluent - usually higher than you would use for another type of tissue).
Hi Maria, Protein block serum free/Dako Ab diluent does improve background staining for CD8 IHC on mouse pancreas, but very tricky. Protein block serum-free block section at RT for 10 minutes, and then incubate CD8 Ab at 37oC for 30 minute which gave best signal vs background result. Other conditions( such as, block longer time(1 hour at RT) , CD8 Ab incubation overnight at 4oC, double CD8 concentration incubation at 37oC for 30 minutes…) cannot give ideal results, either signal much weaker or strong background. Thanks again for your help.
Hi Qian, Thanks for the update. Signal to background at 37oC is difficult to control - I'm glad that you have some decent results. For a long while my preferential method is overnight incubation at 4oC with suitably diluted antibody but I vary the blocking stage and diluents.
Dear Qian,
if you have still background problems, I can offer you a sample of our new polyclonal rabbit Anti-CD8a antibody. It works very well in IHC and IHC-P; here is the link;
http://www.sysy.com/products/cd-8/facts-361003.php
If you are interested, please contact me.
Carsten
Dear Qian,
I am going to do IHC to stain the mouse liver tumors with CD8 antibody in paraffine sections. Does your CD8 staining works well now? Could you share your protocols? Thank you a lot.
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