Evos xl core light microscope

After 21 days culture, the cells were washed with PBS, fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min, washed again and incubated with 40 mM Alizarin Red stain (ARS-pH between 4.1 and 4.5) in ddH₂O for 20 min with gentle shaking on a shaker. ARS was removed, cells were washed five times with ddH₂O and air dried. An EVOS XL Core light microscope (Thermo Fisher Scientific, UK) was used to take images of stained cells at 10× magnification.

After the learned helplessness paradigm, mice were sacrificed, and the brains were recovered to undergo Golgi staining according to the manufacturer’s instructions (FD Neurotechnologies, Inc.). Once fixed, the brains were flash-frozen and 120-μm coronal sections were prepared and mounted onto gelatin-coated glass slides. After allowing the tissue to dry and adhere to the slides for 1 week at 35 °C, samples were dehydrated with sequentially increasing percentage ethanol/ddH₂O solutions and xylene, and finally slides were cover-slipped using Eukitt (#NC9068612, Thermo Fisher Scientific) hardening mounting medium. Hippocampal images were acquired at 100X magnification with the EvosXL Core light microscope (Life Technologies). At Bregma − 2.30 mm, apical dendrites, 20 μm from the neuronal cell body, were imaged within the CA1, CA3, and dentate gyrus anatomical regions of the dorsal hippocampus. Images were analyzed blinded to treatment, and dendritic spine density was quantified using the Neurolucida and Neuroexplorer software, available in the Microscopy Core Facility at the University of Miami, or ImageJ. The average number of dendritic spines over approximately 80 μm of total apical dendrite length (not necessarily from the same neuron) per hippocampal region was determined for each mouse.

Lung tissues were fixed with formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). Inflammatory responses were evaluated by scoring the microscopic findings in 20 visual fields of 12 different sections for each group, using the following criteria regarding the bronchus or bronchial area: 2 for mucus occlusion in the bronchial space, 1 for swelling of alveolar–epithelial cells, infiltration of inflammatory cells in the peri-bronchus, destruction of bronchial epithelial cells, or bleeding in the bronchial space. The following scoring method for the lung parenchyma was employed: 3 for destruction of the alveolar structure of >50%, 2 for that of 25–50%, and 1 for that of <25%, thickening of the alveolar wall, infiltration of inflammatory cells, or bleeding in the alveolar space [32 (link)]. Microscopic images were taken using a Life Technologies EVOS XL Core light microscope at 40× magnification.
Expression of influenza HA antigen was confirmed by staining with monoclonal antibody against influenza H1N1 pandemic HA antigen (ab66189, abcam, Cambridge, UK), goat antibody against mouse IgG conjugated with HRP (ab6789, abcam,), and DAB substrates (Invitrogen, Carlsbad, CA, USA).

To investigate the cell tropism, Vero, A549, and HEp-2 cells were infected with MVAIK, MV/RSV, and RSV (m.o.i. = 0.1). B95a, Jurkat, and U937 cells were also infected in a similar way. Microscopic images to assess the appearance of the cytopathic effect (CPE) were taken using a Life Technologies EVOS XL Core light microscope (Life technologies, USA).

Lung tissues were fixed with 4% formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). Immunostaining was performed using a four-clone blend of monoclonal antibodies against the RSV P, F, and N proteins (AdB Serotec, UK) and anti-mouse IgG conjugated with HRP (Dako North America, Inc.)[17 (link)]. Lung tissue specimens were stained using the Peroxidase Stain DAB Kit and Metal Enhancer DAB stain (Nacalai Tesque, Inc., Kyoto, Japan).
As for the scoring of histological findings, six random fields per group were scored for histopathology using the following criteria: 1, swelling of alveolar walls; 1, destruction of bronchial epithelial cells; 1, peribronchial infiltration of inflammatory cells; 2, mucus as a production cause of occlusion in the bronchial space; and 1, bleeding. Microscopic images were taken with a Life Technologies EVOS XL Core light microscope at 40× magnification.

After the sacrifice of experimental mice, the liver, epididymal white adipose tissue (WAT), kidney, and spleen were isolated to observe possible toxicity and extent of fat deposition in them. Isolated organs were immediately weighed and fixed into 10% neutral formalin solution for 2 days. Fixed tissues/organs were dehydrated in alcohol and embedded in paraffin. Thin tissue slices of tissue of about 5 μm thicknesses were sectioned using a microtome, attached in a slide, and stained with hematoxylin and eosin (H and E). Stained slides were pictured using an EVOS XL core light microscope (Life Technologies, Bothell, WA, USA) at 10x and 20x magnifications.