GATA-3, RORgt staining troubleshooting
I am having trouble getting a good signal for RORgt (151Eu), GATA-3 (152Sm) and CTLA-4 (170Er) antibodies. I stain frozen PBMC (after 2hr resting in media), all three antibodies are stained intracellularly and we do methanol perming before intracellular stain. I tried titrating the antibody to high concentrations and I still don't see good signal. Any suggestions to improve signal? Should I stimulate the cells before staining for these markers? Thank you!