Orca c4742 95 12er charge coupled device camera

For fluorescence microscopy of live yeast to visualize GFP, cells were cultured in SR medium for 18 h at 30 °C, then, the appropriate amount of these cultures was suspended into fresh SG to reach an OD₆₀₀ of 0.3, and they were incubated for additional 4–6 h for GAL1 promoter induction. Cells were harvested by centrifugation and observed directly. To monitor vacuolar morphology and endocytosis, staining with FM4-64 was performed as described [21 (link)]. Nuclear labelling was performed by adding DAPI at 1:1000 directly to the harvested cells in vivo and washed once with PBS. To observe actin, yeast cells were fixed and treated with rhodamine-conjugated phalloidin (Sigma) as described [23 (link)].
Indirect immunofluorescence on yeast cells was performed as previously described [51 (link)]. Antibodies were used as follows: As primary antibody, monoclonal rat anti-alpha-tubulin (Serotec, YOL1/34) at 1:500 dilution; as secondary antibody, Alexa Fluor 594 anti-rat dye (Life Technologies) at 1:1000 dilution. DAPI was added at 1:1000 for nuclear labelling. Cells were examined in Eclipse TE2000U microscope (Nikon, Tokyo, Japan) and digital images were acquired with an Orca C4742-95-12ER charge-coupled-device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and processed by HCImage and ImageJ software.

To monitor PTEN subcellular location in mammalian cells, immunofluorescence was performed as previously described, using mouse monoclonal anti-PTEN 425A and fluorescein-conjugated anti-mouse antibody [18 (link),37 (link)]. For standard microscopy, a Zeiss fluorescence microscope (Thornwood, NY) was used. For confocal microscopy, a Leica confocal microscope (TCS-SP2-AOBS, Mannheim, Germany) was used. For quantitation of PTEN subcellular distribution, at least 100 positive cells were scored for each experiment. Cells were rated as showing nuclear staining (N), cytoplasmic staining (C), or staining within both the nucleus and the cytoplasm (N/C), as illustrated in Fig 1A. Nuclei were identified by Hoescht 33258 (Molecular Probes, Eugene, OR) staining. All pictures were taken under a 400 X magnification. Measurement of green fluorescent protein (GFP)-Akt1 plasma membrane localization in yeast, as an indirect indicator of cellular PIP3 levels, was performed by fluorescence microscopy, as described [36 (link)]. ≥150 cells were examined and scored for each condition or experiment for either cytoplasmic or membrane-associated localization, as illustrated in Fig 1C. Cells were examined under an Eclipse TE2000Umicroscope (Nikon) and digital images were acquired with Orca C4742-95-12ER charge-coupled device camera and HCImage software (Hamamatsu).

For mCherry in vivo fluorescence microscopy, yeast transformants were cultured as usual and cells were collected by centrifugation of 1 mL of culture at 2500 rpm. For actin staining, cells were cultured as usual, fixed with p-formaldehyde, and stained with rhodamine–phalloidin as described previously [39 (link)]. Samples were prepared for visualization and the microscope used was an Eclipse TE2000U (Nikon, Tokyo, Japan) with the appropriate sets of filters. Digital images were acquired with an Orca C4742-95-12ER charge-coupled device camera (Hamamatsu, Hamamatsu city, Japan) and processed with HCImage software (Hamamatsu).