B 150

Animals were selected from the local abattoirs of Narowal city. Animals were sacrificed through the Islamic slaughter method, and after exsanguination, the entire gastrointestinal tract was located and ligated. Both openings of the rumen, esophageal and rumeno-reticulum were ligated, and both dorsal cranial and caudal sacs of the rumen were opened and searched for rumen flukes. Tissue samples of the rumen (1 cm²) were sectioned from animals positive for rumen flukes and then following technique was used as described by Hofmann and Schnorr (1982) and Zitnan et al. (2008) . Collected samples were placed in fixative (neutral buffered formalin) within 2 hr of slaughter. Post-fixation dehydration of tissues was done in ascending grades of alcohols followed by clearing in a xylene solution. Melted paraffin was used to infiltrate the tissues and tissue blocks were prepared in an embedding desk. Tissue blocks were sectioned at 5 μm thickness with a microtome and mounted on glass slides. The tissue slides were then stained with Hematoxylin and Eosin (Suvarna et al., 2019). Stained sections were analyzed under the microscope (B-150, Optika, Italy) for histological changes at 40X and 100X.

From all study groups, organ samples (liver and kidney) were fixed in 10% formalin and embedded in paraffin wax blocks. About 5–7μm thick microtome sections were cut, and H&E dye was used to stain them on a glass slide. Light microscopy (Optika B-150) was used to examine the slides, and Optika Vision Lite 2.1 was used to capture images.

The paraffin-embedded tissue microarray was deparaffinized and rehydrated prior to antigen retrieval. Then, the slide was immersed in the antigen retrieval buffer and heated in the microwave. Soon afterwards, the buffer was allowed to cool to room temperature and was then washed with PBS. Blocking solution was applied to the sections, which were incubated for 1 hour. A primary antibody against Lyn (1:50, Thermo Fisher, USA) was added to the sections prior to incubation at 4°C overnight. The second day, the sections were washed three times with PBS for 5 minutes each. An HRP-conjugated secondary antibody was added to the slide, which was further incubated for 2 hours. A diaminobenzidine (DAB; Beyotime, China) kit was used to observe antibody binding. The microarray was first washed and then stained with hematoxylin, and slides were imaged under a microscope (B-150 OPTIKA, Italy). Cases were scored based on the immunostaining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and percentage of positive cells (0, < 5%; 1, 5%-25%; 2, 25%-50%; 3, 50%-75%; 4, 75%-100%). The final score was calculated by multiplying the above two scores so that the final score ranged from 0 to 12. We defined final scores of 0-4 as “no or low expression” and 5-12 as “high expression”.