Pac1-FRB-GFP Localization by Fluorescence Microscopy

Pac1-FRB-GFP localization was detected by using fluorescence microscopy as previously described (28 (link)). Briefly, liquid cultures were grown in EMM to early log phase (OD600nm 0.3) then rapamycin or an equal volume of DMSO was added to a final concentration of 2.5 μg/ml. After two hours incubation, nuclei were stained using Hoechst 33342 for 15 min (0.2 mg/ml) and live cells were mounted on 1.2% agarose patches. GFP-tagged proteins and nuclei were detected at 470 nm and 365 nm, respectively, using a Colibri system (Carl Zeiss Canada, Toronto, ON, Canada) on a Zeiss Axio Observer Z1 inverted microscope with a ×60/1.4 oil objective. Data were analyzed using the ZEN black software (Carl Zeiss Canada).