Digital light microscope

Fresh liver tissues from each group were fixed in 10% neutral formalin at room temperature for 24 h, dehydrated in ascending grades of ethanol, and embedded in paraffin. Tissue blocks were cut at 4 μm thick. Paraffin sections were stained with hematoxylin and eosin dye and examined using a digital light microscope (Olympus, Tokyo, Japan). The histological scoring was assessed according to López-Alonso [49 (link)]. A minimum of 3 slides for each animal and 10 fields per slide were examined and scored semi-quantitatively for the severity of changes. The scoring was done as none (−), mild (+), moderate (+ +), and severe (+ + +) changes.

C57BL/6 mice (n = 5 mice/group) infected with PR8 virus (as described previously) were administered oral oseltamivir (5 mg/kg) and ivy extract (30 mg/kg) for 2 or 5 days. Lung tissue was rapidly excised from mice sacrificed 6 h after the final treatment. Lung tissue was washed with PBS and fixed in 4% formaldehyde for 1 h at 4°C. The tissue was dehydrated by gradual soaking in alcohol and xylene, and then embedded in paraffin. Paraffin-embedded specimens were cut into 10-μm sections, stained with hematoxylin and eosin (H&E), and viewed with a digital light microscope (Olympus, Tokyo, Japan). A pathologist performed a blind test of the sections under a light microscope and the levels of lung tissue destruction, epithelial cell layer damage, polymorphonuclear cell infiltration, and alveolitis were evaluated for scoring as previously described [19 (link)].

The upper right lobe of the lung was fixed and embedded in paraffin. Specimens were cut into 5-µm sections, stained with hematoxylin-eosin (HE), and observed using a digital light microscope (Olympus, Tokyo, Japan). Vascular hypertrophy parameters were assessed via the ratio of medial artery wall thickness to medial artery outer diameter, and the ratio of the medial artery wall area to the medial artery area.

The regions of mouse jejunum and ileum were washed with PBS and fixed in 4% formaldehyde for 1 h at 4°C. The tissues were dehydrated by gradually soaking them in alcohol and xylene and then embedded in paraffin. The paraffin-embedded specimens were cut into 5 μm sections, stained with hematoxylin-eosin (H&E), and viewed with a digital light microscope (Olympus, Tokyo, Japan). The length of intestine villus was measured by NIH image.

After hemodynamic measurements and blood withdrawals, the hearts and lungs were excised and harvested for fibrosis, morphometric and histologic analysis. The isolated heart and middle lobe of the right lung were fixed in 4% paraformaldehyde for 24 h, then they were embedded in paraffin and sectioned into 5-μm-thick slices. The heart slices were stained with Masson’s trichrome to determine the RV collagen volume fraction, and the lung slices were stained with H and E dyes to determine the arteriole remodeling. Digital light microscope (Olympus, Tokyo, Japan) was used for overall histological assessment. For quantitative analysis, pictures were selected from each section by an investigator who was unaware of the grouping. The fibrosis of RV was assessed by Image-Pro Plus software (Version 6.0, Media Cybernetics, Silver Springs, MD, United States). Pulmonary arterial medial wall thickness (WT) was calculated by the following formula: WT (%) = area_ext-area_int/area_ext × 100, where area_ext and area_int were areas bounded by external and internal elastic lamina, respectively.

HUVECs were grown up to 60–70% confluence in six-well plates. Then, cells were induced by H₂O₂ or/and treated with paeoniflorin or/and treated with EX527 and cultured at 37°C for 24 h. When the confluence was 100%, the cells were scratched by a 1000-μl disposable pipette tip and PBS was used to remove the dead cells. Images were taken by a digital light microscope (Olympus) at 0 h and 24 h after wounding.

Animals on mentioned days were sacrificed by ketamine overdose injection, and the collected tissues were placed in a 10% formalin solution and fixed for 24 h. After that, the samples were put in paraffin blocks, sectioned into 5 µm thick slices using a microtome (Leica Biosystems, Germany), and fixed on coverslips for future staining. The tissue sections were individually stained by Masson’s trichrome (MT) and Hematoxylin and Eosin (H&E) methods. The slides were dehydrated, mounted, and histological photographs were observed and interpreted by a digital light microscope (Olympus, Japan) at ×100 and ×200 magnification. In comparison, angiogenesis, inflammatory cell infiltration, reepithelialization, fibroproliferation, hemorrhage, connective tissue formation, and collagen deposition were assessed comparatively in different groups and on certain days. Histomorphometry analysis was conducted to evaluate the angiogenesis in the healing process.