Coolsnap hq2 ccd camera

Cells were grown on coverslips at 70% confluency and then rinsed in PBS and fixed for 15 min in 4% paraformaldehyde/PBS at room temperature. Fixed cells were washed in PBS and quenched for 10 min in PBS/50 mM glycine, saturated in PBS containing 1 mg ml^−l BSA (blocking buffer) and permeabilized in PBS/0.05% saponin/1 mg ml⁻¹ BSA (incubation buffer, IB). Cells were incubated for 1 h with the primary antibody diluted in IB, washed three times in IB and incubated with the corresponding secondary antibodies diluted in IB for 45 min. Coverslips were washed three times with IB and then mounted in DABCO medium and examined on an Eclipse 80i Upright Microscope (Nikon) equipped with a CoolSNAP HQ2 CCD Camera, a Piezo Flexure Objective Scanner and × 100 Plan Apo objective (1.4 numerical aperture CFI (chrome-free infinity)). Images are maximum-intensity z projections of three-dimensional image stacks acquired every 0.2 μm using the Metamorph software (MDS Analytical Technologies, Sunnyvale, CA).

Transfected neurons were fixed at DIV8 with 4% formaldehyde and 4% sucrose in phosphate-buffered saline (PBS) at room temperature for 10 min. Cells were then washed three times in PBS-CM (PBS, 1 mM MgCl₂, 0.1 mM CaCl₂), permeabilized with 0.2% Triton X-100 for 15 min, and washed one time with PBS-CM, before incubation with 0.2% gelatin for 30 min at 37°C. Next, neurons were incubated with primary antibodies diluted in 0.2% gelatin for 30 min at 37°C, and washed three times in PBS-CM. This was followed by incubation with secondary antibody diluted in 0.2% gelatin for 30 min at 37°C, and washing three times in PBS-CM. Finally, coverslips were mounted in Fluoromount (Invitrogen).
Fixed cells were imaged on: (1) a Nikon Eclipse 80i upright widefield fluorescence microscope, equipped with a Photometrics CoolSNAP HQ2 CCD camera and Nikon NIS Br software, using a Plan Fluor 40× N.A/1.30 oil objective; or (2) a Carl Zeiss LSM 700 confocal laser scanning microscope running ZEN2011 software, using a Plan-Apochromat 40×/1.30 oil DIC objective.

MNT-1 cells grown on coverslips were incubated for 3 days in medium containing peptides (10 µM). Media containing peptides were renewed every day. Coverslips fixed in 100% glacial methanol for 5 s were washed 5 times in distilled water and incubated in PBS/1 mg/mL BSA (incubation buffer, IB). Fixed cells were incubated for 1 h with mouse monoclonal anti–γ-adaptin (Sigma-Aldrich, clone 100/3) and rabbit polyclonal to KIF13A (Bethyl Laboratory, Inc., A301-077A) diluted in IB, washed three times in IB, and incubated with the corresponding secondary antibody (Alexa Fluor, Invitrogen) for 30 min. Cells were washed twice in IB and once in PBS before mounting the coverslips in DABCO medium (Invitrogen) and examining on an Eclipse 80i Upright Microscope (Nikon, Tokyo, Japan) equipped with a CoolSNAP HQ2 CCD Camera, a Piezo Flexure Objective Scanner and 100× Plan Apo objective (1.4 NA CFI).

Spheroids were harvested and frozen in Tissue-Tek® O.C.T., (Miles USA, Inc. Elkhart, IN). Eight μm cryostat sections were obtained with a Leica CM1950 cryotome and transferred on to Superfrost™ Plus slides (Fischer Scientific, Boston, MA) for immunostaining. Slides were incubated in polyclonal rabbit anti-human fibronectin (Q0149, Dako) and collagen Type IV mouse monoclonal antibodies for 30 min at room temperature followed by labelling using a Dako Envision + kit (IR630) from Dako North America Inc. (Carpinteria, CA) according to the manufacturer’s instructions. Signal was visualized with 3,3′-Diaminobenzidine (DAB) followed by a light hematoxylin counterstain. Images were taken at 40X magnification using a Nikon Ti E upright microscope with a Cool SNAP HQ2 CCD camera (Tokyo, Japan) and processed with NIS-Elements basic research software.

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.