Plan apochromat 63 1.40 oil dic

U87 cells (100 000) transduced with Fuw vector or wild-type PK (PK^wt) or GL261 cells (50 000) were plated on 18 mm coverslip previously coated with 0,05 mg/mL of L-poly-L-lysine for 2 hours at 37 °C. Then, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0,1 X Triton-X100 for 10 min. Nonspecific sites were blocked for 20 min with 5% BSA in PBS buffer containing 0,05% Tween-20. Each cover slip was incubated for 2 hours at room temperature with primary antibody (PK MAB 5512 Merck Millipore). A secondary donkey anti-mouse alexa fluor-488 antibody or donkey anti-mouse alexa fluor-594 antibody (Molecular Probes DMS, 1:1000) were used to reveal the primary antibody and the nuclei were stained using DAPI (Molecular probes, 1:15000). Images were acquired either using an inverted laser scanning confocal microscope Leica TCS SP8 3X (Leica Microsystems, Nanterre, France) through a HC PL APO CS2 100X/1.4 Oil immersion objective or with an epifluorescence microscope Axioplan2, Zeiss through a Plan-Apochromat 63×/1.40 oil DIC objective with immersion. Images were organized with OMERO, an open microscopy environment (OME). Final images were obtained after deconvolution using the Huygens Remote Manager version 3.7 (Scientific Volume Imaging, The Netherlands), using the CMLE algorithm, with SNR:45 and 40 iterations.

Confocal images were acquired with a Zeiss LSM 700 microscope using the Plan-Apochromat 63 × 1.40 oil DIC (WD = 0.19) objective. Images were taken using the 405, 488, and 555 nm laser lines. Fluorescence emission of CFW and Hoechst was detected using the 400–490 nm spectral band. Red fluorescence emission of mRFP and dTomato was detected with the 560–700 nm spectral band and FITC fluorescence was detected with the 490–555 nm spectral band. Images were analyzed and processed with the Fiji image processing package of ImageJ¹.

For co‐stainings with rabbit‐anti‐fascin, rabbit‐anti‐p34‐Arc/ARPC2 and mouse‐anti‐EB1, neurons were fixed for 5 min at −20°C in 100% methanol supplemented with 1 mM EGTA, immediately followed by 5 min of fixation at room temperature in 4% paraformaldehyde/4% sucrose. In all other cases, neurons were fixed for 10 min at room temperature in 4% paraformaldehyde/4% sucrose.
After fixation, neurons were washed 2× in PBS. Primary antibodies were diluted in GDB buffer (0.1% BSA, 0.45 M NaCl, 0.3% Triton X‐100, 16.7 mM phosphate buffer, pH 7.4) and incubated overnight at 4°C, followed by 3 × 5 min washing in PSB and 1‐ to 2‐h incubation with secondary antibodies in GDB buffer at room temperature. Samples were mounted in VECTASHIELD mounting medium (Vectorlabs).
Images of fixed cells were collected using (i) a Nikon Eclipse 80i upright widefield fluorescence microscope, equipped with a Photometrics CoolSNAP HQ2 CCD camera and Nikon NIS Br software, using one of the following oil objectives: Plan Apo VC 60× N.A. 1.40, Plan Fluor 40× N.A. 1.30 or Plan Fluor 20× N.A. 0.75; or (ii) using a Carl Zeiss LSM 700 confocal laser scanning microscope running ZEN2011 software and using a Plan‐Apochromat 63×/1.40 Oil DIC objective. For quantitative comparisons between conditions, imaging settings were kept identical for all acquired images.