Cfi plan apochromat lambda 60x oil

Each labeled sample was placed between two glass slides for imaging (VWR rectangular coverglass, 24 × 60 mm) on top of a 60X objective (CFI Plan Apochromat Lambda 60X Oil, NA = 1.4, Nikon, Japan) of an inverted microscope (Nikon Eclipse Ti). A diode laser (λ = 647 nm, Nikon LU-NV Laser Unit) was used as a light source for imaging. Z-stack scanning was realized by employing a 300 nm piezo stepper for a 20 μm z-range. Confocal image generation was performed with a spinning-disk based confocal head (CSU-W1, Yokogawa Electric Corporation, Japan). Image sequences were acquired with a digital camera (Orca-Flash 4.0, Hamamatsu Photonics, Japan).

Cells grown on No. 1.5 coverslips coated with poly-L-lysine were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at 37 °C. Cells were then permeabilized with 0.25% Triton X-100 in PBS for 10 min. After fixation and permeabilization, cells were immunostained as we described previously [25 (link), 26 (link)]. Rabbit monoclonal anti-huntingtin (1:500, D7F7, Cat #5656S, Cell Signaling) was used. Immunofluorescence was detected using a laser confocal microscope (Nikon A1R). DAPI (4′,6-diamidino-2-phenylindole) was used as a nuclear counterstain. Images were taken with a 60X oil objective (CFI Plan Apochromat Lambda 60X Oil, numerical aperture (NA) = 1.4) under a Nikon A1R laser scanning confocal microscope with a Galvano scanner. DAPI signals were imaged using a 405 nm diode laser at 1% power and detected with a photomultiplier tube (PMT). Htt signals were imaged using a 514 nm diode laser at 2.6% power and detected with a GaAsP detector. NIS-elements acquisition software was used to acquire the images with a resolution of 1024 × 1024 pixels.
A Nikon Eclipse Ts2R microscope equipped with a DS-Fi3 was used to obtain brightfield images from WT, B7 and C12 cells with a 20X objective (S Plan Fluor ELWD 20X/0.45). NIS-elements acquisition software was used to acquire the images with a resolution of 1024 × 1024 pixels.

Slides with 30 μm sections from nine larvae per condition were fixed in 4% PFA for 15 min at RT. Sections were washed thrice with PBS and permeabilized with 0.5% Triton (Sigma–Aldrich), washed and blocked with 1% PBS-BSA (bovine serum albumin) (Sigma). Staining was performed with a combination of Bodipy665 (D10001, Thermo Fisher Scientific) diluted at 1/500, Phalloidin (SC-363797, Santa Cruz Biotechnology, Dallas, USA) diluted at 1/2000, WGA FITC (GTX01502, GeneTex, Irvine, USA) at 1/1000, all in PBS-BSA 1% for 2 h at RT then rinsed three times with PBS. Finally, the slides were stained with Hoechst (H3570, Invitrogen Molecular Probes, Eugene, USA) at 1/8000 for 2 min and rinsed thrice. Slides were mounted with Mowiol (Sigma–Aldrich) covered with a 24 × 50 mm coverglass (Merck, Darmstadt, Germany). Images were acquired using a spinning disk confocal microscope (Nikon Ti2, CrEST Optics aX-Light V3, Nikon, Tokyo, Japan) at a 60X magnification in oil immersion (CFI Plan Apochromat Lambda 60X Oil, N.A1.40, W.D. 0.13 mm, Nikon) in stacks of 15 μm thickness with 0.2 μm Z-steps.