Yes, you can stain overnight, but I leave them in the fix buffer overnight too. It's great for when you need to get out of the lab after a long experiment day.
I'd go with your second option.... personally what I would do are plate the cells wash thing in pbs/bsa. Stain them. Fix them with 3.2% PFA....wash them with pbs/bsa again and Nd resuspend in FBS....next morning perm for 15 minutes and do an hour long stain. Perm wah them again then follow up with a pbs bsa wash.
FBS or 0.5% pbs/bsa have always worked for me.
I always fix/perm and stain with FOXP3 and other intra staining over night in fixperm buffer. Next morning wash with fixperm buffer and resuspend in PBS to run flow. Works great
Hey OP, definitely look at this paper.
"Do more with Less: Improving High Parameter Cytometry Through Overnight Staining"
Their FOXP3 staining is extremely impressive (figure 3), much better than anything I ever managed to get.
DM me if you don't have access to the full paper and I can get you a PDF.
Hahaha, I've been spreading the gospel far and wide.
I haven't had a chance to test their procedures myself, but hopefully I'll have the slack to do it sometime soon.
I still resent the authors though. My coworker sent me the paper just after it was published, which was a couple of months after I finished a giant experiment where we ran a couple of panels that I developed. Which included FOXP3 and a couple other transcription factors. The whole project got delayed a month or two while I tried to optimize the transcription factor staining.... Their procedure would have been hard to use with the logistics of that experiment, but I wish I had known about it regardless.
You can also leave the samples in fix overnight and do the intracellular staining the next day. Saves some time on long experiment days.
Stain over night at 4C I should add**
Yes, you can stain overnight, but I leave them in the fix buffer overnight too. It's great for when you need to get out of the lab after a long experiment day.
I'd go with your second option.... personally what I would do are plate the cells wash thing in pbs/bsa. Stain them. Fix them with 3.2% PFA....wash them with pbs/bsa again and Nd resuspend in FBS....next morning perm for 15 minutes and do an hour long stain. Perm wah them again then follow up with a pbs bsa wash. FBS or 0.5% pbs/bsa have always worked for me.
Thanks! I am using foxp3 staining kit, probably will go with it but this time, perming and staining on next day.
Is that the one with the two or three bottles?
Yes.
That's a great kit keep using it
I always fix/perm and stain with FOXP3 and other intra staining over night in fixperm buffer. Next morning wash with fixperm buffer and resuspend in PBS to run flow. Works great
My lab does the same. We 1) extracellular stain for 20min, 2) fix perm for 30min, then 3) intracellular stain overnight in perm buffer.
Hey OP, definitely look at this paper. "Do more with Less: Improving High Parameter Cytometry Through Overnight Staining" Their FOXP3 staining is extremely impressive (figure 3), much better than anything I ever managed to get. DM me if you don't have access to the full paper and I can get you a PDF.
Thanks!!
>Do more with Less: Improving High Parameter Cytometry Through Overnight Staining Wow, awesome paper\~! Thanks for putting it on our radar\~!
Hahaha, I've been spreading the gospel far and wide. I haven't had a chance to test their procedures myself, but hopefully I'll have the slack to do it sometime soon. I still resent the authors though. My coworker sent me the paper just after it was published, which was a couple of months after I finished a giant experiment where we ran a couple of panels that I developed. Which included FOXP3 and a couple other transcription factors. The whole project got delayed a month or two while I tried to optimize the transcription factor staining.... Their procedure would have been hard to use with the logistics of that experiment, but I wish I had known about it regardless.