Excelsior as processor

Routine histological analyses was undertaken. In brief, tissues were preserved in 4% buffered formalin for 24–72 h, transferred to 50% ethanol before being dehydrated through 70%, 96% and absolute ethanol, cleared in xylene and soaked in paraffin wax using an automatic Excelsior AS processor (Thermo Fisher Scientific Inc., USA). Paraffin block sections were cut at 2 μm using a microtome Microm HM 355 S (Thermo Fisher Scientific Inc., USA), dewaxed overnight in at 60 °C and stained with modified Harris haematoxylin and Young’s eosin stains (Thermo Fisher. Scientific Inc., USA). To demonstrate ferric iron in tissue sections, Pearls Prussian blue histochemical method was used (Merck, Germany). After staining, sections were dehydrated in increasing concentrations of ethanol (70–100%), cleared in xylene and mounted in Biomount DPX (Biognost, Croatia). Microphotographs were taken using Axio Scan.Z1 scanning light microscope (Zeiss, Germany) and were edited using ZEN 2.3 imaging software. Severity of histopathological alterations was assessed using semi-quantitative approach as control (0), mild (+), moderate (++) and severe (+++).

Tissue samples were collected in Zinc formalin (Anatech) and fixed for a minimum of 72 hours before being washed and dehydrated using a Thermo Excelsior AS processor. Upon removal from the processor, tissues were transferred to a Thermo Shandon Histocentre 3 embedding station where they were submersed in warm paraffin and allowed to cool into blocks. From these blocks, 5um sections were cut and mounted on charged glass slides, baked overnight at 60°C, and passed through Xylene, graded ethanol, and double distilled water to remove paraffin and rehydrate tissue sections. A Leica Autostainer XL was used to complete the deparaffinization, rehydration and routine hematoxylin and eosin stain. Slides were digitally imaged with a NanoZoomer S360 (Hamamatsu) and subsequently examined by a board-certified, veterinary pathologist using HALO software (Indica Labs).