Ck40 microscope

The histomorphology and the count of goblet cells in the jejunum was determined as described previously [33 (link), 34 (link)]. Briefly, following the fixing, the segment of the jejunum was embedded in paraffin. Consecutive sections (5 μm) were stained with hematoxylin-eosin and Periodic Acid Schiff- Alcian Blue (PAS-AB) for histomorphological examination and counting goblet cells, respectively. The villus height and crypt depth of the jejunal mucosa was measured at 40 × magnification with an Olympus CK 40 microscope (Olympus Optical Company). The number of positively stained goblet cells was counted within 10 randomly selected villi using Image-Pro Plus software, version 6.0 (Media Cybernetics).

The duodenal, jejunal and ileal histomorphology were determined as described previously [34 (link)]. Briefly, following the fixing, the segment of the jejunum was excised, dehydrated and embedded in paraffin. Then, consecutive sections (5 μm) were cut and stained with haematoxylin and eosin. The villus height and crypt depth of the jejunal mucosa were determined by a single experimenter, blind to the pig breed and source of gut microbiota, and measured at 40 × magnification with an Olympus CK 40 microscope (Olympus Optical Company).

After slaughtering, three small intestines (jejunum) of each group were preserved and fixed by immersion in 10% neutral-buffered formalin for subsequent examination. The histomorphology was determined as described previously (34 (link)). Briefly, approximately 5 cm of the middle of the jejunum was fixed, dehydrated, and embedded in paraffin and cut into sections with a thickness of 5 μm for staining with hematoxylin and eosin. The height villus, crypt depth, thickness of muscularis mucosa, thickness of muscle layer, as well as thickness of serous membrane layer were determined by a single experimenter and measured with an Olympus CK 40 microscope (Olympus Optical Company) at × 40 magnification. Each parameter of the 30 portions from five sections were measured, including villus height, crypt depth, muscularis mucosa thickness, muscle layer thickness, as well as serous membrane layer thickness. Six fields in each intestinal section were considered. After measuring the villus height and crypt depth, the ratio of villus height/crypt depth was calculated.

PK-15 cells were cultured in 96-well plates and maintained in DMEM containing 2% FBS. The confluent cell monolayers were respectively infected with HNX-TK⁻/gE⁻-Flt3L or HNX-TK⁻/gE⁻ at 0.05 MOI. After 24 h, PK-15 cells were fixed with ethanol for 30 min at −20 °C, followed by incubation with rabbit anti-Flt3L polyclonal antibody (Bioss) or mouse anti-gB monoclonal antibody (Keqian) for 1 h at 37 °C. After three washes with PBS (Gibco, Thermo Fisher Scientific), the cells were respectively stained with Cy3-conjugated goat anti-rabbit IgG (Abclonal, Wuhan, China) or FITC-conjugated goat anti-mouse IgG (Abclonal) for 30 min at 37 °C. Images were captured using an Olympus CK40 microscope.

Matrigel (BD Biosciences, San Jose, CA, USA) matrix was thawed and maintained on ice at 4 °C; 50 μL of the matrix was transferred to each of the 96-microwell culture plates. The plates were incubated at 37 °C for at least 1 h to allow the matrix solution to solidify before treatment. Aliquots of 200 μL of HUVEC suspended in endothelial cell growth medium with 2.5% FBS (1.5 × 10⁴ cells/well) were placed in the 96-well Matrigel-coated plates (Corning, Glendale, AZ, USA). The vehicle control and DBPR114 were then added to each well in triplicate and incubated at 37 °C for 18 h. Following incubation, the morphology of the endothelial cell tubes in the individual wells was evaluated through photomicroscopy (Olympus CK40 microscope, Tokyo, Japan). The failure of the formation of continuous networks between cell bodies in the presence of drug treatment was photographed and scored depending on the extent of tube disruption at a magnification of × 40. The total tube length in each picture was measured, with ≥ 30% tube formation inhibition relative to the vehicle-treated control group indicating significant anti-angiogenic activity.

To confirm the rescued virus (rPRV-TJ), a swine anti-PRV serum derived from the PRV-TJ strain-infected pigs was used as primary antibody in indirect immunofluorescence analysis. PK-15 cells were seeded in 96-well plates and cultured in DMEM containing 5% FBS. The confluent cell monolayers were infected with serially 10-fold diluted rPRV-TJ for 36 h. The cells were fixed with ethanol for 30 min at -30°C, followed by incubation with swine anti-PRV sera (diluted 1:300 with PBS) for 2 h at 37°C and then with Alexa 488-conjugated goat anti-pig IgG (Thermo Fisher Scientific) (1:1,000) for 1 h at 37°C. Images were captured using an Olympus CK40 microscope.

The morphology in jejunal mucosa was determined as described previously [4 (link)]. In brief, following fixing in 4% paraformaldehyde, the jejunal segment was embedded in paraffin. Then, consecutive sections (5 µm) were stained with hematoxylin–eosin. In each sample, a total of 10 intact villi and crypts were randomly selected, and villus height and crypt depth were measured at 40× magnification with an Olympus CK 40 microscope.