Eclipse e600

The sections were hydrated in PBS for 10 mins and processed for histological staining with hematoxylin and eosin (H&E) following standard procedures. The sections were observed under an Eclipse E600 (Nikon, Shinjuku, Japan) optical microscope and evaluated following the criteria of Gross et al. (48 ). Images were acquired using an optical microscope (Eclipse E600; Nikon, Shinjuku, Japan) equipped with a USB 3.0 camera series “33” Imaging Source (cat. No. DFK 33UX264; Bremen, Germany).

To determine the percentage of eGFP positive cells after infection, sterile glass cover slips coated with Poly-D-Lysine (Sigma-Aldrich) were put in tissue culture flasks prior to cell plating and infection. Seven days after infection, the medium was removed, and cells were washed with PBS and fixed with 4% PFA. Glass cover slips were recovered, placed on microscope slides and, after cell nuclei were counterstained with DAPI, examined for percentage of eGFP positive cells by fluorescence microscope (Eclipse E600, Nikon) in the green fluorescent field.
To examine the percentage of cells with neuronal morphology during the course of retinoic acid and herbimycinA treatment, SK-N-SH cells were grown and treated with differentiation medium in microscope slide chambers. When the treatment procedure was completed, cells were fixed with methanol, stained with Coomassie blue, and examined in the bright light field using Nikon microscope (Eclipse E600, Nikon).

Cells were collected by low-speed centrifugation, washed with phosphate buffer saline (PBS), resuspended in the same buffer, and observed under a microscope (Eclipse E600, Nikon, Japan) at × 400 magnification. Each image taken with the microscope was expanded 5 times and the cell size at the long side of more than 100 cells was measured manually. Acridine orange staining of E. coli cells was performed as follows. Cells were collected, washed with PBS, mixed with 1.0 mg/ml acridine orange solution, and incubated at room temperature for 15 min under a dark condition. Stained cells were visualized by a fluorescent microscope (Eclipse E600, Nikon, Tokyo, Japan) with Nikon B-2A (EX450-490/DM505/BA520) for fluorescein. Intracellularly accumulated ROS were detected using a fluorescence probe, 2’, 7’-dichlorofluorescin diacetate (H₂DCFDA) [16 (link)]. Cells grown until a log phase were mixed with 0.1 mM H₂DCFDA, incubated for 1 h, and centrifuged at a low speed to harvest cells as a pellet. The cells were then washed with PBS, suspended in the same buffer, and disrupted by sonic oscillation. The fluorescence was measured by using a POWERSCAN HT microplate reader (DS Pharma Biomedical) with excitation at 504 nm and emission at 529 nm. Protein concentration was determined by the Lowry method [17 (link)] and was used for normalization.

Liver and kidney tissues from diabetic control mice treated and diabetic mice treated with CA were fixed in 10% neutral buffered formalin for 24 h. After fixation, tissues were dehydrated in a graded alcohol series and, after chloroform treatment, embedded in Paraplast. Deparaplasted 5–6 μm thick sections were stained with hematoxylin and eosin (HE), following standard protocol, and examined under a light microscope (Nikon Eclipse E600, Nikon, Tokyo, Japan). Stained slides were examined under a light microscope (Nikon Eclipse E600) at 100, 200, 400 and 1000× magnification. Liver tissue sections were examined for lymphocyte infiltrations, vacuolization, necrosis and apoptosis. To determine the percentage of apoptotic cells, two hundred cells in randomly selected microscopic fields of vision was examined. Kidney sections were examined for lymphocyte infiltrations, changes in renal corpuscles, renal tubules and apoptosis and necrosis. Photomicrographs were taken by a digital camera (Nikon DMX1200, Nikon, Tokyo, Japan), and imaging software Lucia G 4.80 (Laboratory Imaging Ltd., Prague, Czech Republic).

The brain and cerebellum samples from BALB/c mice infected with the DENV2 NGC or injected with mock were cleaved, fixed in buffered formalin (10%) and blocked in paraffin resin. The tissue sections, cut in 4 µm thick, were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol, as described elsewhere^{62 (link)}. After washing in running water, the tissue sections were stained with hematoxylin and eosin for histological examination and visualized under Nikon ECLIPSE E600 microscope. Quantification of tissue lesions were performed by a semi quantitative analysis in brain and cerebellum sections stained with H.E. For each of the parameters used in the quantification of damages (hemorrhage, perivascular infiltrates and infiltrates in the meninges), a numerical scale ranging from 0 to 4 according to the severity and the extent of damage was assigned (0 = none, 1 = mild, 2 = moderate, 3 = severe and focally, 4 = severe and diffuse). Values were measured covering the entire histological slide, visualized by light microscopy (epifluorescence microscope Nikon ECLIPSE E600).

Incisional punch biopsies of ulcers (3 mm in diameter) were obtained at baseline (pre-treatment) and post-treatment (4 weeks). Microscopic evaluation of routinely haematoxylin & eosin-stained paraffin sections [15 (link),21 (link)] was performed to verify the healing process and images acquired by using a digital camera (E600 Eclipse, Nikon, Tokyo, Japan).