From dnhkng from googlemail.com Mon Nov 3 10:01:28 2008 From: dnhkng from googlemail.com (Dr Mephesto) Date: Mon Nov 3 12:35:16 2008 Subject: [Fluorescent-proteins] DsRed stable transfection doesn't Fluoresce? Message-ID: Hi, I am have trouble with getting stable expression of DsRed in HEK cells. I am using the pStoplight vector which express DsRed until it is cut out by Cre Recombinase, and then GFP is expressed. I have cloned this into the Flip- in kit from Invitrogen to generate a stable cell line using flp recombinase to integrate the vector. It looks good for a while, but then, even though I have cells surviving the selection agent, after a week or so, I dont have any more red fluorescent cells! So, I have no idea whats happening. It seems to me that either: 1) the transient expression of flp recombinase is somehow acting on all the stoplight transfected cells, somehow removing the gene... unlikely. 2) DsRed expression is somehow being driven down by the cell? 3) DsRed is somehow toxic and kills the stable cells, but then why does anything survive!? Anyway, any suggestions or advise would be very much appreciated! From confuseddave from gmail.com Fri Nov 21 05:55:09 2008 From: confuseddave from gmail.com (Confused Dave) Date: Fri Nov 21 09:06:26 2008 Subject: [Fluorescent-proteins] Quenching Fluorescent Proteins Message-ID: Hello, I'm looking for a way to reliably inactivate fluorescent proteins (EGFP and dsRed) without harming epitopes for immuno. A little background: I'm electroporating inducible constructs into the chick embryo. We use dsRed as the electroporation control, and EGFP as a marker for activation of our doxicyclin-inducible construct. We want to use immunofluorescence to on these samples. We currently use 4% formalin to fix, washes with PBS+tween, usually ethanol dehydration for storage, and sucrose-agar embedding for sectioning, and the fluorescence still comes up strong. Obviously, imaging with epifluorescence with a green and red channel, we can't add another fluorophore. Best case would be a way to specifically kill the RFP leaving the green intact, although as long as we can recover the GFP with an antibody, this isn't a problem. Also looking for something that won't damage our epitopes to badly! Thanks, Dave