Eclipse ni u optical microscope

Whole-Fish: Fixed larvae were washed with phosphate buffered saline (PBS) for 5 min, and then penetrated by 20, 40, 80 and 100% 1,2-propanediol (Sigma, United States) each for 15 min. Then stained with 0.5% Oil Red O (Sigma, United States) at 65°C in the dark for 1 h. The zebrafish larvae were then incubated in 100% 1,2-propanediol at room temperature for 1 h, and eluted with 80, 40, and 20% 1,2-propanediol each for 10 min. Finally, larvae were imaged by an Olympus U-HGLGPS microscope (Tokyo, Japan).
Cryosections: Fixed zebrafish larvae were dehydrated in 30% sucrose at 4°C for 3 days. After being embedded in optimal cutting tissue (OCT) compound (Leica, Germany), larvae were cut into 14 μM sections. Frozen sections were washed with PBS to remove OCT, incubated in 100% 1, two propylene glycol for 5 min, then stained with 0.7% Oil Red O for 10 min at 60°C, finally eluted with 85% 1,2-propylene glycol and rinsed with PBS to keep the background clean. The slices were imaged with a Nikon Eclipse Ni-U optical microscope (Nikon, Tokyo, Japan).
Cell slides: Fixed cell slides were washed with PBS for 15 min, then incubated with 100% 1,2-propanediol for 10 min. After being stained with 0.7% Oil Red O and decontaminated with 85% 1,2-propanediol and PBS, the slides were imaged with Nikon Eclipse Ni-U optical microscope (Nikon, Tokyo, Japan).

Lung tissues were fixed with freshly prepared zinc formalin for 7 days, moved to 75% ethanol for dehydration, embedded in paraffin at the melting point of 55 °C, and cut to 5-µm sections per sample. The sections were baked at 60 °C for 2 h, and de-waxed in xylene for 10 min and then rehydrated by placing into a decreasing concentration series of graded ethanol solutions and distilled water, followed by staining with hematoxylin and eosin. For immunohistochemical analysis, lung sections were heat-treated for epitope retrieval in a pressure cooker with citrate buffer (pH 6.0), and incubated with freshly prepared 3% hydrogen peroxide for 30 minutes to block endogenous peroxidases, followed by incubation with blocking buffer. The slides were incubated with a rabbit anti-SARS-CoV-nucleoprotein polyclonal antibody (Novus, Cat#NB100–56576) at 1:1000 dilution overnight at 4 °C, and then incubated with a 1:2000 dilution of HRP-conjugated goat anti-rabbit IgG (Abcam, Cat#ab205718) at ambient temperature for 45 min. Subsequently, diaminobenzidine was added to the lung sections, followed by counterstaining with hematoxylin, and then rinsed in distilled water. The microscopic images were captured using a Nikon Eclipse Ni-U optical microscope.

Growth was determined from cell quantification throughout culture monitoring to define the exponential division rate per day (μ, d^-1) as per [ref. 11 (link)]. Cell quantification was determined using a haemocytometer (Neubauer Haemocytometer, Fisher Scientific, Loughborough, UK) as per [ref. 87 (link)]. Average cell volume for each isolate was determined from a minimum of 50 cells from images captured using NIS-Elements AR software (v.4.30.000) and an Eclipse Ni-U optical microscope coupled with a DS-Fi2 colour camera (Nikon, Tokyo, Japan). Images were processed on the NIS-Elements AR software to calculate spherical volume from cell diameter, of all cells present in each image. Elemental measurements were subsequently considered relative to both division rate and volume as both can influence nutrient acquisition [65 ].