Jess, Cayman Chemical has a FITC conjugated Ab that works. You'll need to do good blocking to get it to work, though. I recommend 2 blocking steps: 1) Dilute the Ab in 5% nonfat dry milk and incubate 30 min at 37 degrees before using. 2) Resuspend the permeabilized cells for 30 minutes in 5% nonfat dry milk before staining. I've used BDs perm solution with good success in this protocol. Fix first with mathanol free formaldehyde (Polysciences), then permeabilize with BD perm, then resuspend in 100 uL of the 5% milk solution and incubate 30 min at rt. Then add your Ab straight into this suspension. This worked well on LPS stimulated human monocytes. Without the milk blocking steps, the background is outrageous. This should work well with saponin as well, just add a step with 5% milk in saponin staining buffer before adding the Ab. Good luck. ---------------------- Keith Bahjat, Ph.D. Scientist, Cancer Vaccines Cerus Corporation Concord, California ----- Original Message ----- From: "Jess Jankowsky" <jankowsky.1@osu.edu> To: cyto-inbox Sent: Friday, November 01, 2002 1:21 PM Subject: cox-2 antibody > > We are interested at looking at COX-2 in a human cell line. Does anybody > have any recommendations for a fluorochrome conjugated antibody that they > know works? Also would you be willing to share your protocol? > > thanks > > Jess >
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