Hm340e

The tissue specimens were immersed in formalin for 24 hours, and then embedded in paraffin and sectioned (paraffin microtome, HM340E, Thermo FIsher Scientific). After deparaffinization and antigen retrieval, the slides were blocked with 10% normal goat serum for 30 minutes at room temperature, and incubated with anti-HIF1α (36169T, Cell Signaling Technology) antibody solution for 24 hours at 4°C with gentle shaking. The images were scanned with a pathology section scanner (Pannoramic MIDI) after DAB staining. For dual immunofluorescent staining, mouse anti-CD31 (GB12063, Servicebio) antibody was incubated with rabbit anti–α-SMA (GB111364, Servicebio), anti–claudin 5 (GB11290, Servicebio), and anti-desmin (GB11081, Servicebio) antibody solution for 24 hours at 4°C with gentle shaking. The images were scanned after incubation with mouse-derived (red) and rabbit-derived (green) fluorescent secondary antibodies. The staining intensity was analyzed with ImageJ (NIH).

After the autopsy, the small intestines (duodenum, jejunum, and ileum) were fixed in 10% formalin and embedded in paraffin according to standard procedures. Formalin-fixed paraffin-embedded tissue blocks were sectioned into 5–8 um thicknesses using a standard rotary microtome (HM-340E; Thermo Fisher, Waltham, MA, USA) and placed on slides. The tissues were deparaffinized using xylene and treated in ethanol (100%, 90%, 70%, and 50%) for 5 min each. The tissue sections were then stained with hematoxylin and eosin (H&E) for histopathology. For immunochemistry, antigen retrieval was conducted using citrate buffer (pH 6.0) at 95 °C for 30 min and at room temperature for 20 min. Subsequently, the sections were incubated with anti-PEDV monoclonal antibody (Median diagnostic, Chuncheon-si, Republic of Korea) overnight at 4 °C. The samples were then labeled with horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G antibodies (Vector Laboratories, Newark, CA, USA) and developed using the 3,3′-diaminobenzidine (DAB; Vector Laboratories, Newark, CA, USA) according to the manufacturer’s instructions. The samples were counterstained with methyl-green, and all slides were visualized and imaged using a light microscope (BX53, DP80; Olympus, Tokyo, Japan).

Histological examination was performed as described previously (Pei et al., 2021 (link)). Tissues collected from the spleen, liver, head kidney, and intestines of control and XX2021-injected fish were fixed with 4% paraformaldehyde for 12 h, submerged in 70% alcohol, dehydrated in tertiary butyl alcohol (10%–100%), and embedded in paraffin. Thereafter, paraffin-embedded tissues were cut into 5-μm-thick sections using a microtome (Thermo HM340E, American) and then stained with hematoxylin and eosin. The histological slides were examined with a light microscope (Olympus BX51, Japan).

The tissues were fixed in 10% phosphate-buffered formalin, routinely processed and then embedded in paraffin. Tissue Sections (6 μm) were prepared using a microtome (HM-340E, Thermo Fisher Scientific, MA, USA), placed on a glass slide and stained with haematoxylin and eosin (H&E). H&E staining was performed following standard procedures. With 6 μm-thick paraffin sections, histopathological examination in the frontal, parietal and occipital lobes of the cerebrum, the cerebellum, brain stem (mid brain, pons and medulla oblongata) and spinal cord (cervical, thoracic and lumbar) was performed.

Twenty-four-week-old male C57BL/6 mice (n = 5) were deeply anesthetized with diethyl ether and sodium pentobarbital (30 mg/kg) and were transcardially perfused through the left ventricle with heparin (10 U/ml) in saline, followed by fixation with 4% paraformaldehyde in phosphate-buffered saline (PBS). After perfusion, the mandible containing the teeth and PDL was removed from the skull. The specimens were immersed in the same fixative for 2 hours at 4 °C and were then decalcified in Kalkitox solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 5 days. The specimens were then embedded in paraffin. Paraffin blocks were cut into 6-μm-thick sections using a microtome (HM340E; Thermo Scientific, Waltham, MA, USA) in the axial and coronal planes. The sections were stained with haematoxylin and eosin and observed.