I have been testing new CD4, CD19 and IL-17 monoclonal antibodies for IHC that were made to work on formalin-fixed paraffin sections. These antibodies are from eBioscience and work really well in lymphoid organs such as the spleen, thymus and LN in mice. However, in neural tissue they seem to bind to myelin unexpectedly. Has anyone come across this problem before and are there any recommendations to avoid the myelin staining but still detect the specific lymphocyte staining? Thanks
Hi Maria,
The fact that your stain worked in lymphoid organ is that because such organs are rich in T cells which naturally lacks the FC receptor and here there is no chance of non specific binding but in your neural tissue it is worth to consider the cellular composition and understand their receptors . If any cell own fc, you need to use blocking reagents such as BSA or any other blocker ideal to your optimized protocol. I hope it helps. Good luck.
Hello Maria,
I would also recommend that you optimise your blocking step. Try different blocking reagents, I think you should block with serum (1-10% solutions) preferably from the same species as the secondary antibody, you can also try BSA or casein as previously suggested. Furthermore you can optimise the length and conditions for your blocking step; overnight at 4oC or 30 mins - 2 hours at room temperature.
Also, do you know what species your antibody was raised in? If it is the same species as your sample then you should expect a lot of non-specific binding by your secondary antibody. In that case you will need to block with a F(ab) fragment before incubating with your primary antibody.
Lastly, using TBS-T buffer instead of PBS-T also helps to reducing background staining.
I hope these tips will be helpful.
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