Hello Estelle,

You could try the fixation protocol from this link:

http://mcb.berkeley.edu/labs/koshland/Protocols/MICROSCOPY/gfpfix.html

Commonly, when you want to image fluorescent proteins in fixed cells it is best to use mild fixation in 3.7%/4% paraformaldehyde for a short period of time in order to preserve the localization of the protein of interest.

Thank you Jana. I also found that one and I think i will try. I was just wondering about the need of a mounting medium and so on ? Do you think cells can be directly observed after this protocol ? And what is the sonication for ?

I fix E. coli cells for FISH in 3.7% PFA in pH 7.4 PBS; room temperature; 30 minutes; wash with PBS. After further treatment for FISH, the YFP (Venus) and mCherry signals are maintained and match live-cell observations for the same cells. Probably this will work for yeast as well. I've also fixed E. coli samples with mEos2 (GFP to RFP photoswitching) with the same protocol but without the subsequent FISH steps.

For yeast and E. coli, I image both live and fixed cells on gel pads made with 3% agarose in minimal/low-fluorescence growth media or PBS. In the past, I've used poly-L-lysine coated coverslips. I use very clean coverslips for single molecule experiments, but this might not be necessary. You might want to check your live samples on the confocal microscope first; if the expression level is low, increased photobleaching in confocal microscopy can be a problem. Depending on your confocal system and the localization of your fluorescent protein you need a minimum of ~1 to 10 thousand molecules per cell.

Sonication is to break up clumps of yeast cells. I find this isn't necessary if I take cells from a shaking culture.

Thank you very much Zach. :-) Now I am considering to do FISH in my strains expressing GFP and a RFP (probably mRuby2), so it is very good to know that your Venus and mCherry signal are maintained.

I was a bit worry about my FISH protocol for yeast that includes an incubation (or even long-term storage) in 70% Ethanol (after fixation and before hybridization with FISH probes) because I know that Methanol fixation for example is very bad for GFP. Does your protocol include such kind of step?

Another of my concern is the compatibility of mounting mediums and anti-fade reagents with fluorescent protein signals. According to the labeling of the FISH probes that I want to use, I would like to use GLOX anti-fade buffer, but I have not been able to find any information about the effect of this buffer on FPs. What mounting medium did you use in your experiments ?

Again, many thanks for your help.

I fix cells with GFP by formaldehyde, 20 minutes with 1.8-3.5% doesnt seem to ruin much of the signal (im working with S. cerevisiae yeast)

Zach Hensel can you describe the agarose gel pads in more detail? I am seeking a better way to immobilize the yeast for imaging in multichannel experiments. Do you bond the yeast to the agarose somehow?