Ipp 6

The cell monolayer was scratched using a sterile 10-μL pipette tip and washed with PBS to remove detached cells. The remaining cells were cultured in serum-free medium, and photos were taken at 0 and 36 h. Gap widths were measured using IPP 6.0 software (Media Cybernetics, Bethesda, MD, USA), and data acquired from three areas of the wound on each plate were used to calculate the mean gap width at a given time.

The total protein was extracted from placental tissues and HTR-8/SVneo cells using RIPA lysate (Beyotime, China) (with PMSF-protease and phosphatase inhibitor added), and then the protein concentration was measured by the BCA assay kit (Beyotime, China) according to the instructions of manufacturer. The protein samples of 30ug per well were added to 10% of the twelve-alkyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slot to separate the protein and then electrotransferred onto the PVDF membranes. Next, the nonspecific site of the membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 ℃ with first antibodies NLRP3 (1:1000, ab214185, Abcam), Caspase-1 (1:500, ab62698, Abcam), GSDMD (1:500, sc-81,868, Santa Cruz) and β-actin (1:1000, AF0003, Beyotime). On the second day, the membranes were incubated with the horseradish peroxidase (HRP) labeled secondary antibodies (1:1000, A0208/A0216, Beyotime) at room temperature for 1 h. Finally, under the action of ECL luminescent liquid, immunoreactive signals were detected by the Bio-Rad gel imaging system (MG8600 Thmorgan Biotechnology Co., Ltd., Beijing, China). The IPP6.0 software (Media Cybernetics, Singapore) was used to analyze the gray values and calculate the relative protein levels of NLRP3, Caspase-1 and GSDMD. The experiment was repeated at least three times.

TUNEL was used for in situ detection of apoptosis in the liver and pancreas. Tissues were paraffin dewaxed to water, washed in phosphate buffered saline (PBS), pH 7.4 twice for 5 min each time, then incubated in 3% hydrogen peroxide for 20 minutes. Samples were then PBS washed twice more and dried. Reaction solution (TUNEL kits, Roche Diagnostics GmbH, Germany) was added to each sample (50 µl), and the reactions were performed at 37°C for 60 min in the dark. After incubation, samples were again washed twice in PBS and 50 µl Converter-POD (TUNEL kits, Roche Diagnostics GmbH, Germany) was added to the sample and incubated at 37°C for 30 min. Samples were washed again in PBS and a DAB Horseradish Peroxidase Color Development Kit (Beyotime Institute of Biotechnology, Shanghai China) was used to stain the samples. Hematoxylin was used to stain the nuclei. Samples then underwent conventional dehydration and were transparently mounted with glycerol. Colored nuclei were read as positive cells. The specimens were analyzed using a computerized image analysis system (IPP6.0 software, Media Cybernetics Inc, USA), and the data were represented as the number of TUNEL-positive cells per field (at 400X).

Paraffin-embedded sections were stained with CD68 protein adducts using a polyclonal antibody (1:100 dilution; Zhong Shan-Golden Bridge Biological Technology Co. Beijing, China) to observe quantitative expression levels of CD68.
The specimens were analyzed using a computerized image analysis system (IPP6.0 software, Media Cybernetics Inc., USA). Two independent observers were blinded to treatment groups when analyzing CD68 staining. The cells where the cytoplasm stained brown, as observed under an optical microscope, were defined as positive cells. The hepatocytes and macrophages were calculated from 8-10 fields, and the percent of positive cells was calculated as followed: number of CD68 positive cells / number of total hepatocytes counted. (at 400X)

Pigmentation spots on the body surface of all ducks were photographed under the same conditions. First, the digital camera was set to manual exposure, and each image was collected under identical exposure conditions, including exposure time and aperture. Then, the obtained images were imported into IPP 6.0 software (Media Cybernetics, USA) and magnified by the same multiplication factor. Using an irregular shape tool incorporated in the software, the area of interest (AOI) of each pigmentation spot was selected, and the geometric size of each region was measured. Pigmentation spot areas on the dorsal and ventral sides of ducks were measured. The proportion of pigmentation distribution on the body surface for each duck was calculated. Three replicates were made for each measurement, and the average values were taken as the final phenotype. A total of 300 ducks were selected to measure the pigmentation phenotypes at 4 and 8 weeks old.

Serial sections of the aortic valve were stained with oil red O and en face of the proximal aorta was stained with Sudan IV as previously reported (Luo et al., 2015b (link)). Briefly, obtained hearts were embedded with OCT, and serial 8-μm-thick frozen sections with 40-μm intervals were obtained and stained with oil red O. The images of plaque size and lesion area were analyzed using IPP 6.0 software (Media Cybernetics, Rockville, MD, United States).