Paraformaldehyde (pfa)

The cells were seeded into a 96-well plate for 24 h. The solution was removed, and monolayers of cells were washed twice with PBS. Then, 10-fold series dilution of viral samples was added to the 96-well plate. Each group was diluted in 8 duplicate wells and cultured at 37°C for 3–5 days. Immunofluorescence assay was conducted to observe and record PEDV-infected cells. Briefly, cells were fixed with 4% paraformaldehyde (Dalian Meilun Biotech Co., Ltd., Dalian) for 15 min and then permeabilized with 0.2% Triton X-100 (Dalian Meilun Biotech Co., Ltd., Dalian) for 10 min at room temperature. After the permeabilization treatment, cells were blocked with 1% bovine serum albumin (BSA) (Beijing Solarbio Science and Technology Co., Ltd., Beijing) and then stained overnight at 4°C with anti-PEDV N polyclonal antibody diluted with 1:10000 (Xu, et al., 2020 (link)). After being washed with PBST three times, cells were incubated with goat anti-mouse secondary antibody (Abcam, Shanghai) diluted with 1:10000 labeled with fluorescein isothiocyanate (FITC) at 37°C for 1 h. The fluorescent particles were observed and counted using a fluorescence microscope (Leica DMi8, Germany).

The mice were euthanized by CO₂ from compressed gas cylinders. Then the tissues were removed and fixed in 4% paraformaldehyde (Meilunbio, China) for more than 24 h, and embedded in paraffin. The sections (thickness, 6 μm) were stained with hematoxylin & eosin or Periodic acid-Schiff (PAS) solutions (Servicebio, China). The tissues injury scores and the number of PAS-positive cells per unit of length (mm) of the basement membrane were determined in 5 randomly selected nonoverlapping fields from respective individual tissue sections using a microscope (DMi8; Leica) and all histology analyses for H&E staining were conducted in a blinded manner as a combination of tissue damage (score 0–5) and inflammatory cell infiltration (score 0–5) with a total ranging from 0 to 10. The number of PAS-positive cells per unit of length (mm) of the basement membrane were assessed with ImageJ software version 1.52 (National Institutes of Health software, Maryland, USA).

Logarithmically growing cells of interest were collected and seeded into 6-well plates (500 cells per well) and cultured in an incubator for 14 days. After the incubator culture was completed, the original medium was discarded. The 4% paraformaldehyde (Meilunbio, China), 0.1% crystal violet staining solution (Meilunbio, China) and PBS were used to fix, stain, and wash. Subsequently, the 6-well plate was placed in a fume hood to dry. Colonies larger than 50 cells were counted. Three independent experiments were performed in each experimental group.