Eclipse te2000 e fluorescence microscope

Cortical neurons (DIV 7-12) were treated with vehicle or NMDA (150 µM) in the absence or presence of G3 or G4 phosphorous dendrimers at the indicated concentrations for different lengths of time. Afterward, the cells were incubated in K–H solution containing the ROS-sensitive fluorescent dye chloro-methyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA 10 µM; Molecular Probes, Barcelona, Spain) or with the mitochondrial-specific superoxide-sensitive fluorescent dye MitoSOX Red (2.5 µM; Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C. After being washed twice with K–H solution, the coverslips were placed on the stage of a Nikon Eclipse TE2000-E fluorescence microscope (Nikon, Tokyo, Japan). Excitation and emission wavelengths were set at 535 nm and 635 nm for H2DCFDA fluorescence and 510 nm and 580 nm for MitoSOX Red, respectively. Samples were recorded every 15 s using a CCD camera (Hamamatsu Photonics, Shizuoka, Japan) and analyzed using the NIS Elements AR software (Nikkon, Tokyo, Japan). Recorded fluorescence for each experimental condition was fitted to the equation y = a + bx, and the slope b was taken as an index of the rate of superoxide production as previously described [57 (link)].

The bacteria were visualized by FISH and 4,6-diamidino-2-phenylindole (DAPI) staining using a 60x Plan Apo oil objective in conjunction with an optional 1.5x multiplier lens on an Eclipse TE2000-E fluorescence microscope (Nikon Instruments Inc., Melville NY) and photographed with a CoolSnap EZ camera (Photometrics, Tuscon, AZ) controlled by MetaMorph software (Nashville, TN). Quantification was only performed when the hybridization signals were strong and could clearly distinguish intact bacteria morphologically by either 2-color (universal and bacterial specific FISH probe) or 3-color (FISH probes and DAPI stain) identification. A minimum of 4 different endoscopic biopsy specimens/organs were evaluated for their mucosal bacterial content. Bacterial quantification was performed in 10 representative fields at a final observed magnification of 600x or 900x. The ten fields included bacteria found within 4 well-defined mucosal compartments: (1) bacteria contained within the mucosa, (2) bacteria attached to the surface epithelium, (3) bacteria localized within adherent mucus, and (4) bacteria found within free mucus.

Tissues were processed, hybridized with FISH probes, stained with 4,6-diamidino-2-phenylindole (DAPI), imaged, and counted as previously described [55 (link)]. The Eubacteria probe (Eub338) has been previous described, and the E. coli probe sequence was 5’-GCAAAGGTATTAACTTTACTCCC-3’ [55 (link)]. Briefly, sections of proximal colon with intact contents were fixed overnight in formalin before moving to 70% ethanol and paraffin embedded. 3-μM thick sections were deparaffinized, labeled with probes overnight, and stained with DAPI. Tissues were imaged with x60 Plan Apo oil objective in conjunction with an optional x1.5 multiplier lens on an Eclipse TE2000-E fluorescence microscope (Nikon Instruments Inc., Minato, Tokyo Japan). A CoolSnap EZ camera (Photometrics, Tuscon AZ) with MetaMorph software (Nashville, TN) was used to photograph images. Counting was performed as previously described with 8–10 sections of each tissue in the lumenal, attached, and invasion compartments [55 (link)].

Paraffin-embedded tissue sections were used to perform immunofluorescence staining. First, the sections were incubated in Triton X (Sigma, Munich, Germany) at room temperature for 15 min and then blocked with 3% BSA (Applicher, Darmstadt, Germany) for 1 h. The sections were then incubated with primary antibodies at 4 °C overnight (TH; Millipore, Burlington, VT, USA), Iba1, and Muc2. Next, the sections were washed 3 times and incubated with the secondary antibodies for 1 h at room temperature. Finally, the sections were washed, embedded with a coverslip, and stored in the dark. A Nikon Eclipse TE2000-E fluorescence microscope was used to capture images at 20× magnification.

Cells were seeded and treated on coverslips. They were then fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 in PBS for 10 minutes, washed twice with PBS, and blocked with 1% BSA in PBS for 1 hour, cells were incubated with primary antibodies diluted in blocking buffer. Primary antibodies used for immunofluorescence as follows. Mouse anti-integrin αvβ3 (LM609, Millipore), mouse anti-laminin-5 (D4B5, Millipore) and rat anti-integrin α6 (GoH3, eBioscience). Secondary antibodies were donkey anti-mouse IgG FITC conjugated (GE healthcare) and donkey anti-rat IgG FITC conjugated (GE healthcare). Coverslips were mounted in Prolong Gold with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Fluorescence images were taken by Nikon Eclipse TE2000E fluorescence microscope (Nikon, Tokyo, Japan) with digital camera.

Fluorescent signals from C6 and U87 cells were recorded as previously described [61 (link)]. Cells were seeded at a concentration of 100,000 cells/mL onto 20 mm diameter glass coverslips (Deckglässer; Baden-Württemberg, Germany) and cultured in 6 well plates (Sardstedt; Nümbrecht, Germany) for 24 h in DMEM supplemented with 10% heat-inactivated fetal calf serum; 2 mM l-glutamine, 5 μg/mL streptomycin, and 20 units/mL penicillin maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Afterwards, cells were treated with the rhodamine-labeled generation 4 bis-MPA dendron (1 µM) in culture medium for 4 h. The cells were then washed three times with Krebs–Henseleit solution with the following ionic composition (in mM): NaCl, 140; CaCl₂, 2.5; MgCl₂, 1; KCl, 5; HEPES, 5, Glucose, 11; (pH: 7.4) and mounted on the stage of a Nikon Eclipse TE2000-E fluorescence microscope (Nikon, Tokyo, Japan). The samples containing the cells were stimulated and recorded at the following wavelenghts (excitation/emission): rhodamine B G4 dendron (530 nm/600 nm), FAM-siRNA (488 nm/520 nm), and Hoescht (350 nm/450 nm), and observed through a 40x oil immersion objective. Data was obtained using the NIS Elements AR software (Nikon, Tokyo, Japan).

The mitochondrial transmembrane potential (Ψm) was determined as previously described [58 (link)]. Briefly, cortical neurons (DIV 7-12) were treated with either vehicle or NMDA (150 µM) in the absence or presence of G3 or G4 phosphorous dendrimers at the indicated concentrations for different lengths of time. Afterward, the cells were incubated in K–H solution containing tetramethylrhodamine methyl ester (10 µM; TMRM) (Thermo Fisher, Madrid, Spain), washed with K–H solution, and mounted on the stage of a Nikon Eclipse TE2000-E fluorescence microscope (Nikon, Tokyo, Japan). The cells were excited at 535 nm wavelength and emitted fluorescence recorded at 590 nm using the NIS Elements AR software (Nikkon, Tokyo, Japan). Samples were recorded every 15 s for 5 min with a CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Decaying fluorescence signal showing mitochondrial potential was fitted using a linear regression model and the least squares method. The slopes of the fitted lines were taken as the rate of loss of Ψm. Percentages of Ψm loss rates were calculated with respect to vehicle-treated cells.