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GATA-3, RORgt staining troubleshooting

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akshayar

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Posts: 1

Joined: Thu Jul 28, 2022 11:28 pm

Post Fri Jul 29, 2022 9:05 pm

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!
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jlederer

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Posts: 10

Joined: Thu Feb 08, 2018 2:42 pm

Post Mon Aug 01, 2022 4:14 pm

Re: GATA-3, RORgt staining troubleshooting

Like many labs, we have tested a number of reagents to optimize intracellular and nuclear staining by CyTOF Abs. We settled on using eBioscience buffer reagents for intracellular and intranuclear staining protocols. We also use heparin to block non-specific staining. You can download our intracellular staining method from our website to see how we stain for these markers. https://ledererlab.bwh.harvard.edu/cytof-core/ Hope this helps.

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