Hi Flow Folks,

I've got a puzzle I need your help with. I've developed a flow assay to stain for intracellular neuronal markers. The protocol uses either 2%PFA fix with 0.1% Triton X-100 perm or the Thermo eBioscience FOXP3 kit. With either kit I also add 10% donkey serum to the perm/wash staining buffer. I also maintain the perm condition through the primary and secondary stains since I'm using indirect staining methods. I've used this method to titrate a total of 6 unique intracellular antibodies. I have good staining index for these markers.

Here's where it get interesting. To test the antibody specificity I want to use negative control cells that do not express the markers of interest. Using Human Protein Atlas I selected BJ, PC-3 and Reh cell lines. However, when I run this protocol I get positive staining on my negative cells! This has me quite puzzled because we have validated these antibodies for use in ICC and know that they localize correctly. Take a look at the attached image for one marker (MAFB) to see what I'm talking about. Secondary only and also isotype plus secondary give negative staining so I don't believe this is just simply blocking. What do you think?

-Mike