Dear all,
I transformed the fresh water ciliate Tetrahymena termophila with GFP. The transformation was successful, as they conferred resistance to a selective antibiotic.
The expression is inducible with Cadmium chloride (at 1ug/ml). When I did fluorescence microscopy, I got no signal in living cells, but a visible signal in Glutaraldehyde fixed cells. I am now wondering why. Has someone experienced similar things? Or can someone explain why?
The signal in the fixed cells is relatively weak (1sec exposure time), I thought about increasing the Cadmium concentration. But I don't know if it actually would help something..
Thank you!
Cheers
When aldehydes react with endogenous tisue amines, the products are strongly fluorescent in the GFP channel; glutaraldehyde is particularly bad for inducing strong autofluorescence.
This can be alleviated somewhat by fixing in paraformaldehyde, but some autofluorescence will still be present. I would recommend taking this step first, then attempting to separate autofluorescence signals from any that your GFP may be producing, if indeed it is.
Best of luck
Thank you Richard! I did some more microscopy on Tuesday and figured the same thing out. It felt kind of stupid,
Anyway Yesterday I went back to the microscope and tried some new "fixing" i strategies. First I tried to place them on small agar pads, which worked but gave quite a bit of background, In the end figured that the best way is to mix the cultures 1:1 with 100% Glycerol. It worked perfectly!
Anyway thank you for your help!
Simon I have the same problems with Tetrahymena and I have to ask if you always induce it with Cadmium. My GFP-protein fussion is under the promoter of an endogenous gene, so I thought it wouldn't be necessary to add Cadmium apart from the inductor of the gene... but I had no expression (checked by WB). When I added CdCl I had both signal on WB and fluorescence in living cells, but still had difficulties in seeing fluorescence on paraformaldehyde- fixed cells. And, if I mix 1:1 cells with 100% glycerol, they expand and deform!
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