Images plus 3

The left lung tissue was placed in a tube filled with 4% paraformaldehyde (Servicebio, Wuhan, China, G1101), followed by conventional paraffin embedding. Paraffin-embedded sections were made. Hematoxylin-eosin staining (HE) was used to observe the morphological changes in lung tissue of mice, and Masson staining was used to observe the collagen deposition. The pictures were detected by a microscope (Motic, BA410E, Motic China group CO., LTD. China) equipped with Motic images plus 3.0 (Motic, Motic China group CO., LTD. China). The image was magnified at 200 ×, with a resolution of 683 × 705, horizontal and vertical resolutions of 96dpi, and a bit depth of 24.

The dimensions of 50 randomly selected sporangia and 50 gametangia (oogonia and antheridia) were measured from the isolates PhF66 and PhF66–. Microscopic examinations and measurements were performed as in Jung et al. [54 (link),55 (link)], except that the sporangia for measurements were produced as in the sporulation experiment 3. Characteristics of mature gametangia (antheridia and oogonia) were examined on clarified VA after 28–35 days at 20 °C in the dark. Abortion rates were estimated from three plates per isolate by counting 100 oogonia in two 15 × 15 mm squares, one cut from the area closer to the edge, and the other from the area closer to the center of the plate. Microscopic measurements of all structures at ×400 were made using an optical microscope (Motic© BA410E), camera (Moticam 5 + 5.0 MP), and camera software (Motic© Images Plus 3.0). The photographs were taken using a compound microscope (Zeiss Axioimager.Z2, Carl Zeiss AG, Oberkochen, Germany), a digital camera (Zeiss Axiocam ICc5), and biometric software (Zeiss ZEN). Statistical differences were obtained by Bonferroni corrected two sample t-test in R (v 3.6.3).

The apparent density of PUR composite was determined according to EN ISO 845 [41 ] standards. For this purpose, samples with dimensions 50 × 50 × 50 mm³ were prepared and measured with a thickness gauge (accuracy ± 0.01 mm) and weighted on analytical balance (accuracy ± 0.001 g).
The cellular structure was determined by using scanning electron microscope (SEM) and X-ray tomography techniques. SEM analyses were conducted with the use of an SU3500 Hitachi microscope (Hitachi, Tokyo, Japan). The cell sizes of the PUR composite were determined using Motic Images Plus 3.0 software (Motic, Hongkong, China).
In order to evaluate the dispersion of wood particles in the PUR composites, the X-ray tomography method was used. For this purpose, samples with dimensions 50 × 50 × 50 mm³ were scanned with the use of a Hyperion X9Pro tomography (MyRay, Italy). The resolution and lamp voltage were 0.3 mm and 90 kV, respectively.

To measure the effect of the ADBR2-blockade on ROS production in 786-O and ccRCC primary cultures, the DCF-DA solution reagent was used (ROS0300, OzBiosciences, San Diego, CA, USA).
Briefly, 5 × 10³ cells/well were seeded in a 96-well plate and incubated in complete medium with [0–50 μM for 786-O and 0–100 μM for ccRCC] propranolol or ICI. After 72 h, cells were washed with PBS and then incubated with 100 μL DCF-DA for 30 min at 37 °C in darkness. After DCF-DA removal, 100 μL/well PBS was added and fluorescence (exc: 485 nm/em: 535 nm) was measured using a GLOMAX multi-detection system (Promega). Fluorescence signal was normalized to cell number.
Bright field and fluorescence microscopy images from the same samples were taken using a Pantera microscope and the Motic Images Plus 3.0 software (Motic, Wetzlar, Germany). FIJI-Image J software tool (NIH, Bethesda, MD, USA) was used to process and quantify the fluorescence intensities.

H. pylori colonization was evaluated according to the number of gastric glands containing bacteria and their density, as follows: 0: No observed bacteria; 1: Occasional pits and/or glands with individual bacteria; 2: frequent pits and/or glands with individual bacteria; 3, infrequent pits and/or gland with dense bacterial colonies; and 4, frequent pits and/or glands with dense bacterial colonies (22 (link)). Sections were stained with hematoxylin and eosin (H&E) for histopathological evaluations of the mouse gastric mucosa describing inflammation, infiltration, metaplasia, and anatomical localization of the lesions, using published guidelines (23 (link)): 0, no infiltration, 1, patchy or multifocal small islands of inflammatory cells in the mucosa and/or submucosa; 2, coalescing aggregates of inflammatory cells in submucosa or mucosa; 3, organizing nodules of lymphocytes and other inflammatory cells in submucosa and mucosa; 4, follicles and/or sheets of inflammatory cells extending into or through muscularis propria  adventitia. Photomicrographs were captured using the bright field microscope Motic BA400 and Motic Images Plus 3.0 software (China).

FFPE bladder tumors and healthy tissue sections were screened by immunohistochemistry for CD44 and Tn and STn antigens, as previously described by Peixoto, A et al. 31 (link). Tn antigen expression was evaluated using the biotinylated VVA lectin (Vector Laboratories, 40 mg/mL, 1 hour at 37 ºC; Table S2) and the detection of STn and CD44 antigens were performed using the anti-tag-72 (B72.3 + CC49; Abcam, 0.5 mg/mL, overnight at 4 ºC; Table S2) and anti-CD44 (1:5000, ab157107, Abcam; Table S2) antibodies, respectively. Lack of cross-reactivity of VVA for blood group A and AB antigens was confirmed using the anti-blood group A monoclonal antibody (HE-193, Thermo Fisher, 1:5, overnight at 4 ºC; Table S2). Sialidase treatment of tissue samples prior to anti-STn probing was also performed to confirm the presence of the glycan (Sigma-Aldrich, 0.2 mg/mL, overnight at 37 ºC). CD44 and anti-tag-72 were detected using Novolink Polymer Detection System (Leica) according to manufacturer guidelines. Biotinylated VVA was detected using Streptavidin, Horseradish Peroxidase Conjugate (Thermo Fisher, ready-to-use, 30 min, RT) followed by incubation with ImmPACT® DAB Substrate, Peroxidase (Vector, 30:1000, 5 min, RT). All images were acquired on a Motic BA310E microscope (Motic) using the Motic Images Plus 3.0 software (Motic).

The migration assay was conducted by suspending human fibroblasts in a culture medium at a concentration of 4 × 10⁵ cells/mL. By using the chamber of the cell culture insert (ibidi, Munich, Germany), an amount of 70 μL of the cell suspension was added into each gap. Following a 24 h incubation period, the cell culture insert was removed, creating a defined cell-free space of 500 μm. The wells were then washed with PBS to remove any potential cellular remnants. Thereafter, punicalagin or ellagic acid was added at doses of 10⁻⁶ or 10⁻⁷ M, and the cells were then incubated under standard culture conditions. Images were captured at 0, 4, 8, 12, and 24 h after treatment using inverted phase contrast microscopy. Motic Images Plus 3.0 software (Motic, Hong Kong) was employed to quantify cell migration areas. The percentage of gap closure was determined using the following formula:
where W₀ represents the initial width of the space immediately following the culture insert assay, and W_n denotes the width at various measurement time points. This formula provided a quantitative measure of the reduction in the cell-free space over the specified time intervals, facilitating a comprehensive analysis of the impact of punicalagin and ellagic acid on human fibroblast migration.