Ds fi1

For histological analysis, the samples were kept for 24 h in 10% formaldehyde. After fixation, the material was submitted to dehydration followed by paraffin embedding, and 4 μm thick histological sections were stained with hematoxylin-eosin, for general morphology studies.
The following scores were used for organ evaluation: Chiu score was used for intestinal evaluation10 (link), Scheuer score for hepatic evaluation11 (link), ventilator-induced lung injury score for pulmonary evaluation12 (link), and a modified Banff score for renal evaluation13 (link).
The histomorphometric analysis consisted of measuring the height and the diameter of the villi, the depth of crypts, the thickness of lamina propria, and the thickness of the muscle layer of the intestine.
The samples were analyzed with a Nikon Eclipse 50i optic microscope. Ten fields per sample were randomly selected and analyzed with a DS-Fi1 camera, using NIS-Elements software version 4.6 to acquire images. The different variables were quantified, and the results were transferred to an electronic spreadsheet, showing an average of the measures.

During necropsy, the inferior lung lobe was inflated with 10% formalin/PBS and placed into 10% formalin/PBS. Following routine processing for paraffin-embedding, 5 μm sections were stained with hematoxylin and eosin (H&E) and assessed for degree of lung involvement by a veterinary pathologist blinded to the experimental groupings using a defined scoring system adapted from Dormans et al. [36 (link)]. The score was based on the total number of lesions, overall extent of changes, presence of peribronchiolitis, perivasculitis, alveolitis, “granuloma" formation and degree of necrosis. The scale ranges from zero (no apparent changes) to five (severe changes). Microphotographs were captured using a Nikon Eclipse 51E microscope equipped with a Nikon DS-Fi1 camera with a DS-U2 unit and NIS elements F software. Microphotographs are reproduced without manipulation other than cropping and adjustment of light intensity.

All fluorescence microscopy was performed on a Nikon Eclipse 90i with a Nikon DS-FI1 color camera, using NIS Elements software. Animals were washed off the plates with M9 buffer and paralyzed in 1 mM levamisole in a microfuge tube. The suspended animals were briefly centrifuged, and 2 µL of paralyzed animals were placed on an agar pad. Animals were aligned with a hair pick and photographed with a 10X Nikon CFI Plan Fluor. GFP excitement range was 450–490 nm and emission was 500–550 nm, while mCherry excitation was from 528–553 nm and emission was 590–650 nm. Pseudocolorization was added linearly by NIS Elements, and images were not altered any further.