Eclipse te2000 u inverted

Imaging experiments were done in Tyrode’s buffer (as described above). Tyrode’s solution containing either 0, 2, or 8 mM Ca²⁺ with osmolarity titrated to 310–320 mosM, and 50 μM APV and 10 µM CNQX to prevent recurrent neuronal activity. Fluorescence was recorded using a Nikon Eclipse TE2000-U inverted microscope equipped with a ×60 Plan Fluor objective (Nikon, Minato, Tokyo, Japan), a Lambda-DG4 illumination system (Sutter Instruments, Novato, CA, USA) with FITC excitation and emission filters, and an Andor iXon + back illuminated EMCCD camera (Model no. DU-897E-CSO-#BV; Andor Technology, Belfast, UK). Images were acquired at 50 Hz to resolve fast spiking glutamatergic peaks. To induce photobleaching, the neutral density filter within the LAMDA-DG4 illumination system was removed in order to use 100% light intensity. This filter was reintroduced for subsequent live imaging of neurotransmisison after photobleaching.
Spontaneous activity was recorded over the course of 6–10 min. Evoked responses were elicited using a parallel bipolar electrode, delivering 35 mA pulses (0.1ms duration) at 5-s intervals. At the end of each experiment, presynaptic boutons were visualized by delivering a high-frequency electrical stimulation (25 Hz 20 action potentials) or by perfusing 90 mM KCl in Tyrode’s solution.

Neutrophils mounted on microscope slides and THP-1 derived macrophages growing on coverslips were incubated with anti-phospho-p40phox antibody (Sigma) followed by Alexa 568 conjugated secondary antibody. Stained cells were analyzed using a Nikon Eclipse TE2000-U inverted microscope with a UV filter set. Intensity scan was created using an Ultraview Program (Perkin Elmer, Waltham, MA).

Skin lesions were embedded in paraffin, sectioned (4 mm thick), deparaffinised in xylene and rehydrated in a gradient of alcohol solutions. The sections were stained with haematoxylin-eosin (H&E). The H&E-stained slides were visualised using an Eclipse TE2000U inverted microscope with twin CCD cameras (magnification, ×200; Nikon).
Abramov’s histological scoring system was used to score epithelisation, fibrosis, angiogenesis and collagen levels67 (link)68 (link). This system assigns a score of 0–3 for each parameter. Inflammation (the number of macrophages) was scored as follows: 0–25 = 1, 26–50 = 2 and >51 = 3. Fibroplasia was graded as follows: 0 (none to minimal fibroblasts), 1 (a few fibroblasts), 2 (more fibroblasts) and 3 (predominantly fibroblasts). Angiogenesis was graded as follows: 0 (none), 1 (up to five vessels per high-power field [HPF]), 2 (6–10 vessels per HPF) and 3 (more than ten vessels per HPF). Epithelisation was graded as follows: 0 (none), 1 (partial), 2 (complete, but immature or thin) and 3 (complete and mature).

Erythrocytes infected with P. falciparum 3D7 parasites were washed twice with Ringer’s solution (122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 0.8 mM MgCl₂, 11 mM D-glucose, 25 mM HEPES, 1 mM NaH₂PO₄, pH 7.4) and incubated with either 10 µM H-Leu-NHMec or 10 µM H-Arg-NHMec for 10 min in the presence or absence of 50 µM bestatin. As a control, parasites were also incubated with the N-terminally-blocked N-carbobenzyloxy (Z)-Leu-Arg-NHMec that is not cleaved by either aminopeptidase. Parasites were imaged on glass slides using an Imager M2 Axio (Carl Zeiss GmbH) with a Plan-Apochromat 100×/1.4 oil objective, an ET-DAPI 49000 filter set and a Hamamatsu C10600-10B ORCA-R² camera or in an open chamber using a NIKON Eclipse TE2000-U inverted epifluorescence microscope with a Plan-Apochromat 100×/1.4 oil objective and a Hamamatsu 1394 ORCA-ERA camera.
Images of at least ten individual parasites were taken per treatment using the same microscope settings and the same solutions under physiological conditions. The relative fluorescence intensities within the parasite cytosol were compared with an area void of cells in the DIC channel using ImageJ 1.53c. The fluorescence intensities within the respective regions of interest in the DAPI channel were then used to calculate mean background-corrected fluorescence intensities.

The protocol for live cell imaging has been reported before.^{13 (link)} Hippocampal neurons were transfected with constructs in a Nucleofector device (Amaxa Biosystems) and were cultured in glass-bottomed 35-mm dishes (Willco Wells) for 3 days. The performance of neurons was obtained using an Eclipse TE 2000-U inverted fluorescence microscope (Nikon, Shanghai, China) and observed with a motorized Z drive using a × 63 oil-immersion objective lens (1.0 NA). Images of the cells selected for imaging were acquired every 1 s continuously for 1 min. The vesicle was handled and processed by the Meta-Morph software. A kymograph was generated by using NIH ImageJ.