Clsm 510 meta

C2C12 myoblasts were seeded on round coverslides (13 mm diameter) and induced to differentiate as previously [4 (link)]. After CisPt treatment cell culture was fixed for 20 min in 4% paraformaldehyde in PBS and permeabilized for 5 min with 0.1% Triton X-100. To block nonspecific binding, slides were incubated in 10% BSA in PBS for 1 h at room temperature. Successive slides were exposed to rabbit polyclonal LC3 (1 : 200) or mouse monoclonal alpha-tubulin (1 : 1000, clone B-512) primary antibodies and then to Alexa Fluor-568 goat anti-rabbit IgG or CY3 anti-mouse secondary antibodies for 1 h (1 : 400). To counterstain nuclei we used DAPI at 5 μg/mL for 10 min at room temperature. Slides were mounted with Prolong Gold antifade reagent (Molecular Probes) and maintained in a cold chamber in the dark until observation. For immunofluorescence imaging we used a confocal light microscope (CLSM 510 Meta, Zeiss) equipped with 488–568 nm laser lights.

Reporter gene expression was visualized using confocal laser scanning microscopy (cLSM-510META, Carl Zeiss, Jena, Germany) using a Plan-Neofluar 40×/1.3 Oil DIC objective. The eGFP fusion proteins were excited with an argon laser at 488 nm with 8–10% of laser capacity. Excited fluorophores were detected with a bandpass filter GFP (505–530 nm) using a photomultiplier. Chlorophyll a autofluorescence was simultaneously detected with a META-channel (644–719 nm). MitoTraker Orange CM-H₂TMRos (Molecular Probes) was applied for fluorescence staining of mitochondria. P. tricornutum cells were stained according to the protocol of the manufacturer. Cells were incubated with 100 nM dye solution, incubated for 30 min, washed and observed (images were recorded using the Multitracking mode with the following parameters for Wavelength T1 = 488 nm 10% and T2 = 543 nm 100% laser line, primary beam splitting mirrors UV/488/543/633 nm; emitted light was detected with the META-channel).