Haematoxylin eosin

Alternate sections were deparaffinized, rehydrated and stained with haematoxylin-eosin (Bio Optica, Milan, Italy) according to the standard procedure. The sections were then observed using a light optical microscope (Olympus BX50 microscope, Hamburg, Germany). According to Guidotti et al. [44 (link)], a blind examiner identified MD, BD and MT hepatocytes using the Olympus BX50 microscope at a final magnification of 1000×. The number of all polyploid hepatocytes was evaluated in 10 random fields in each liver sample.

After the experiment, brain tissue was removed, fixed at room temperature in buffered formaldehyde solution (10% in phosphate buffered saline), dehydrated by graduated ethanol, and then embedded in paraffin. Light microscopy was used to examine tissue sections that were 7 um thick after being deparaffinized with xylene and stained with haematoxylin/eosin (Bio-Optica, Milan, Italy). The number of damaged neurons was counted, and the grey matter’s histopathologic alterations were graded on a 6-point scale: No lesion was found, 1; 1–5 eosinophilic neurons were present in the Gray matter, 2; 5–10 eosinophilic neurons were present, 3; more than 10 eosinophilic neurons were present, 4; a small infarction (less than one third of the grey matter area), 5; a moderate infarction (one third to one half of the Gray matter area); and 6, a large infarction (more than half of the grey matter area). To determine a final score for each mouse, the results from every part of each brain were averaged. The slices were then analysed by a blinded histopathologist using an optical microscope using a Leica DM6 microscope (Leica Microsystems Spa, Milan, Italy) (Petrosino et al., 2017 (link)).