Fv1200 laser scanning microscope

Cells grown on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were stained with freshly prepared ~50 μg/mL of filipin (a fluorescent dye that specifically binds to cellular free cholesterol) in PBS for 1 h at room temperature. Stained cells were imaged using an Olympus FV1200 laser-scanning microscope equipped with four detectors and six laser lines (405, 458, 488, 515, 559, and 635 nm). Laser line 405 nm was used to detect filipin signals and 559 nm was used to detect mCherry fluorescence. The manufacturer’s software and Fiji software were used for data acquisition and analysis.

The yeast cells were cultured to the mid-logarithmic growth phase in SD/-Met liquid medium. The cultures were centrifuged at 5000 rpm for 2 min and resuspended in a fresh medium containing 80 μM FM4-64 (Invitrogen) for vacuoles staining. Yeasts were centrifuged after 1 h of incubation, resuspended in the fresh medium again, and cultured for extra 3 h. Yeast cells were then sampled on glass slides and observed under Olympus FV1200 Laser Scanning Microscope.

Larval brains were imaged on the ventral side. Fixed imaging of larval brains was performed using an inverted Olympus FV3000 Laser Scanning Microscope with Becker and Hickel FLIM system and with an inverted Olympus FV1200 Laser Scanning Microscope with high sensitivity galium arsenide phosphide (GaAsP) detectors (Olympus). Images were acquired using ×20 0.75 NA UPlanSApo, ×40 1.3 NA Oil UPlan FLN, ×60 1.4 NA and ×100 1.4 NA Oil UPlanSApo objective lenses. The laser units used were solid state 405 and 488 lasers, argon 488, 515, 568 and 633 lasers.

LMNA⁺, KD and Control cells were seeded on cover-glass supports in complete medium and allowed to grow up to 70% confluence. To study Lamin A/C expression, the cells were fixed in absolute methanol for 5 min at −20 °C. Non-specific binding was blocked with 5% non-fat dry milk in PBS. Lamin A/C was detected using an anti-Lamin A/C monoclonal antibody (Jol2; Chemicon). Alexa Fluor 594 goat anti-mouse was used as a secondary antibody. For F-actin localization the cells were fixed in 2% paraformaldehyde in PBS containing 2% sucrose for 30 min, were permeabilized in PBS containing 0.2% Tween-20 and were blocked with 5% non-fat dry milk in PBS. Cells were then incubated for 30 min with 5 μg/mL TRITC-phalloidin (Sigma) in PBS. In both cases the nuclei were stained with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 min in PBS. Finally, the cells were washed in PBS and briefly rinsed in double distilled H₂O and glass coverslips were mounted in ProLong Gold anti-Fade Reagent (Molecular Probes). Images were acquired using a confocal laser scanning microscope (Olympus FV1200 Laser Scanning Microscope). The brightness and contrast of the acquired images were adjusted, and the figures were generated using Adobe Photoshop CS3 (Version 10.0; San Jose, CA, USA).

For GFP tagging of NbSWP12, DNA fragment of Nbswp12 was amplified by PCR from p-Cold I-Nbswp12 plasmid with primers containing BamH I or Sal I restriction site (Table 1) and inserted into pUG35 plasmid. The pUG35 and pUG35-Nbswp12 plasmids were transformed into yeast S. cerevisiae CEN.PK2 using Yeastmaker™ Yeast Transformation System 2 (Clontech, Mountain View, CA, USA). Transformants were selected in Minimal synthetic defined (SD) bases with Uracil Dropout Supplement (SD/-Ura). Isolated clone was grown in SD/-Ura liquid overnight and inoculated into SD/-Ura-Met inducing medium for overexpression under control of MET25 inducible promoter. Yeasts were collected at 24 or 48 h post inoculating, and location signals were observed under Olympus FV1200 Laser Scanning Microscope.

Mesenchyme explants were prepared and cultured as described above in media containing 0.1 µg/mL Hoechst dye solution (Thermo Fisher Scientific) used to label all nuclei. Chamber slides containing attached explants were imaged at 3 min and 27 s intervals for 17 hr using an FV1200 laser scanning microscope (Olympus) with a stage top incubator (Tokai Hit). Movies were generated using Imaris Software (Oxford Instruments).

The previously generated strain Rluv3-Gal4, UAS-EB1::GFP (Yalgin et al., 2015 (link)) was used to investigate the organization of MT minus ends in D. melanogaster class I da sensory neurons. Preparation of embryos was carried out as previously described (Yalgin et al., 2015 (link)). Briefly, stage-15 embryos were quickly de-chorionated, washed in water, and mounted with halocarbon oil with one layer of double-sided tape as a spacer. Imaging of D. melanogaster was performed with a FV1200 Laser Scanning Microscope (Olympus) equipped with a 473 nm laser, and a PLAPON 60XO NA1.42 (Olympus) objective. Time-lapse images were acquired at 5% laser power at 5x zoom, imaging three z-sections spaced 0.6 µms apart, with 2.4 s between frames, for four minutes. Kymographs were prepared using ImageJ using an average projection of imaged z-sections.