Plan apo 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).

One hour after electroconvulsive stimulation (ECS), rats were deeply anesthetized with an overdose of pentobarbital solution, and removed brains were frozen in crushed dry ice and coronal sections of 14 μm thickness were cut on a cryostat and attached to a slide glass. Sections were fixed in 4 % paraformaldehyde and then incubated in PBS containing 4 % donkey serum (Chemicon, Temecula, CA) and 0.2 % Triton X-100 for 1 h at room temperature. Slices were incubated with rabbit c-fos antibody (1:500, sc-52, Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and mouse anti-NeuN antibody (1:500, MAB377, Chemicon) overnight at 4 °C. After washing with PBS, the slices were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:300, Molecular Probes, Eugene, OR), and Alexa Fluor 546-conjugated donkey anti-mouse IgG (1:300, Molecular Probes) for 2 h at room temperature. ProLong Gold Antifade Reagent (Molecular Probes) was used to protect samples from bleaching. Fluorescence images were obtained using the all-in-one fluorescence microscope, BZ-9000 (Keyence, Japan) through a 4× Plan-Apo objective (Nikon, Japan).

Cells were plated at a density of 50,000 cells/well in six-well plates on coverslips and allowed to attach overnight. Media was removed and cells were treated with/without GANT61 (20 μM) for 48 h. Coverslips were removed and placed in a humidity chamber for fixation by absolute methanol for 10 min at 4°C. Cells were permeabilized with acetone for 1 min at 4°C. After washing with PBS × 3, cells were incubated with the diluted primary antibody overnight at 4°C. Cells were subsequently washed x 3 and incubated with appropriate secondary antibody at room temperature in the dark for 1 h. After washing with PBS, cell nuclei were stained with DAPI at RT for 5–10 min. Confocal images were acquired on a Nikon A1 laser confocal system with a Nikon Eclipse Ti microscope and a 60X Plan Apo objective. Lasers used were 405 nm for blue, 488 nm for green, and 561 nm for red. NIS Elements AR 4.5000 software was used to acquire Z-stacks of each channel sequentially to avoid spectral cross talk. Each slice was captured at a 0.15 μm step. Primary antibodies used were γH2AX (Millipore 1:200), NBS1 (Novus 1:200), and MRE11 (Cell Signaling 1:200). The co-localization index for specific foci and fluorescent intensity were calculated by ImageJ.

Fluorescence images were obtained using a motorized inverted wide-field epifluorescence microscope (Nikon Eclipse Ti-E), using Nikon 10× Plan Apo objective (N.A. = 0.4). Motorized excitation and emission filter wheels (Ludl Electronics) fitted with a DAPI/CFP/YFP/DsRed quad filter set (#86010, Chroma) were used together with filter cubes for DAPI, YFP, and TxRed (Chroma) to select specific fluorescence signals.

A 1:1 mixture of 2500 A375 cells (RFP positive) and SK-Mel28 cells (no fluorescent protein) were seeded together in 96-well plates and treated under the same conditions as the Zebrafish cells except now low serum conditions were at (0.5%) and high (5%).
Using a GE INCell 6000, 2 fields were captured per well using a Nikon 10 × Plan Apo objective, 0.45NA. Images of the Hoechst signal was captured via the 4,6-diamidino-2-phenylindole channel with 0.1 and 0.3 seconds exposure in order to identify the nucleus and cell body, respectively. The dsRed channel used for RFP, with a 2-second exposure. Nuclei and cell bodies were identified as per the Zebrafish cells. Cells were classified as either RFP positive (A375; having an average intensity within the nuclear mask of greater than 283 in the dsRed channel) or negative (SK-Mel28). Measurements were captured for all cells as well as RFP positive and non-fluorescent cells separately.