Digital ccd camera

Whole mount preparation of dissected gonads and immunostaining procedures were performed as in Colaiácovo et al. (2003) (link). Immunofluorescence images were captured with an IX-70 microscope (Olympus, Waltham, MA) fitted with a cooled CCD camera (CH350; Roper Scientific, Tuscon, AZ) driven by the Delta Vision system (Applied Precision, Pittsburgh, PA). Images were deconvolved using the SoftWorx 3.0 deconvolution software from Applied Precision. Flourescent images showing RHO-1 expression and dpMPK-1 expression were captured with a Zeiss Axioskope microscope fitted with a Hamamatsu digital CCD camera.

Purified human platelets (1x10⁷ platelets/sample) treated with various concentrations of minocycline for 30 minutes at 37°C were incubated on glass coverslips coated with fibrinogen (150 μg/mL; Sigma Aldrich, Saint Louis, MO, USA) for 45 minutes and blocked with 0.5 μg/mL BSA (Sigma Aldrich, Saint Louis, MO, USA). Platelets were subsequently fixed with 4% paraformeldahyde and washed with PBS. The platelets were stained with phalloidin-Alexa Fluor 488 at a 1:200 dilution in PBS with 0.01% Triton for 45 minutes at room temperature. The coverslips were then mounted on slides and platelet spreading was imaged with a 20x objective using a Ziess Axiovert 200 fluorescent microscope (Ziess, Thornwood, NY, USA) coupled with a Digital CCD Camera (Hamamatsu Photonics, Hamamatsu City, Japan). Images were processed using ImagePro Software (Media Cybernetics, Rockville, MD, USA) and spreading was assessed and quantified based on of platelet morphology and categorized as: 1) fully spread (indicative of lamellopodia), 2) partially spread (indicative of filopodia, but not lamellopodia), or 3) not spread (retaining its discoid shape).

Fifty thousand cells were plated per well of an 8 well μ-slide microscopy chamber (ibidi) 24 hours before treatment. Following drug treatments, cells were fixed with 4 % paraformaldehyde in PBS for 15 minutes, permeabilized with 0.3 % Triton X-100 in PBS for 10 minutes and blocked with 3 % BSA in 0.3 % Triton X-100/PBS for 1 hour. Cells were then incubated with primary antibodies against LAMP1 (Hybridoma Bank; #H4A3-s), TFEB (Cell Signaling; #4240S), or p-4E-BP1 (Cell Signaling; #2855S) in 0.3 % Triton X-100/PBS at 4 °C overnight. Fluorescence staining was performed using anti-rabbit Alexa Fluor 488 or 594 secondary antibodies (Life Technologies; #A11008, #A11012) in 0.3 % Triton X-100/PBS at room temperature for 1 hour. Fluorescence microscopy was performed with a DeltaVision microscope system (Applied Precision) using a 60x oil immersion objective (Olympus) and a digital CCD camera (Hamamatsu Photonics). Following acquisition, images were deconvolved with Softworks V3.5.1 (Applied Precision) to increase spatial resolution. Images were prepared using ImageJ (rsbweb.nih.gov/ij/). Representative images shown are total intensity projections (Z-axis scans).

Calcium imaging was conducted as previously described by Joseph and colleagues [16 (link)]. Briefly, following DA exposure, hippocampal neurons were incubated with Fura-2/acetoxymethyl ester (2 µM) in loading media (Neurobasal Media) for 40 min (37 °C, 5% CO₂), followed by a 30 min incubation in Krebs-Ringer buffer (0.3 mM CaCl; 131 mM NaCl; 1.3 mM MgSO₄; 5.0 mM KCl; 0.4 mM KH₂PO; 6.0 mM glucose; 20 mM HEPES; pH 7.4). Real-time analyses of calcium flux were conducted in 8-well chamber slides mounted on the stage of a Nikon Eclipse TE2000 inverted fluorescence microscope coupled to a digital CCD camera (Hamamatsu Photonics, Bridgewater, NJ, USA) and illuminated with a fluorescent light source. Simultaneous images of cells at λex 340/380 nm and λem 510 nm were captured at 5 s intervals using Elements software (Nikon) to control a MAC 2000 filter/shutter controller (Ludl Electronic Products, Hawthorne, NY, USA). After approximately 45 s, cells were depolarized by adding 30 mM KCl and image capture continued for 10 min total. Pixel-by-pixel comparisons of the captured images were conducted to generate a ratio of Ca²⁺-bound Fura (340 nm excitation wavelength) to unbound Fura (380 nm excitation wavelength) for each pair of images. Intracellular calcium ([Ca²⁺]_i) was determined using the method of Grynkiewicz and colleagues [23 (link)].

We used the membrane permeability indicator Fura-2AM (Dojindo, Kumamoto, Japan) to assess changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of isolated GCs as described previously [15 ]. Fluorescence images were captured on an Olympus inverted microscope equipped with a digital CCD camera (Hamamatsu Photonics, Shizuoka, Japan). We used high-speed continuous scanning monochromatic light sources (Till Photonics, Grafeling, Germany) to excite at 340 nm and 380 nm. Fluorescence intensities at 340 nm and 380 nm (F₃₄₀ and F₃₈₀) were measured every 1–10 s, and images were acquired using C-imaging systems (Hamamatsu Photonic). The [Ca²⁺]_i of the cell was proportional to the ratio of fluorescence intensity between the two images. Before an experiment, we measured the background fluorescence level and subtracted it from the obtained data.

In each animal, an entire series of brain sections (1:4 or 1:8), containing the whole SNpc or the whole DVC, were stained using cresyl violet (CV) to identify key anatomical structures and structural integrity. Brain slices in representative groups were stained for TH as described below. TH-positive neurons in the SNpc were quantified using the Stereo Investigator software suite from MBF bioscience with a 100x magnification using a Olympus BX53 microscope (Olympus, Tokyo, Japan) fitted with a digital CCD camera (Hamamatsu, Hamamatsu City, Japan) and a motorized stage (Prior Scientific, Rockland, MA, USA). The total numbers of cells were estimated using the optical fractionator,^{60 (link)} the coefficient of error was calculated according to Gundersen et al.,^{61 (link)} and values ≤0.05 were accepted as significant. TH stained sections were counterstained with CV to assess neuronal loss, as opposed to TH down-regulation, and estimated independently using design based stereology as detailed above.