Hi, I am currently optimizing the staining for CD137 on mouse HCC/spleen (also known as 4-1BB) using Opal immunofluorescence system. My blocking steps include (1) 5% non-fat milk for 30 mins (2) peroxidase blocking for 10 mins and (3) TMB blocking for 30 mins. The results showed that my isotype control and unstained control are both clean without any unspecific staining. However, the samples treated with anti-mouse CD137 antibody displayed high background, though some cells appeared to have significantly stronger signals.

So to solve this problem, I have tried to (1) lower my antibody concentration from 5 to 1 ug/ml and (2) switch opal 520 to opal 570 fluorophores. Yet the problems still persist.

Can anyone provide suggestions for improvement of my staining?Thank you