Hematoxylin eosin staining kit

Mouse testis and epididymis were fixed in 4% paraformaldehyde at 4 °C for 24–48 h, embedded in paraffin wax, and sectioned at 5 μm. The sections were then deparaffinized and rehydrated as follows: Incubation in xylene for 20 min (performed twice), followed by successive incubations in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and ddH₂O for 2 min each. HE staining was carried out using the Hematoxylin-Eosin (HE) Staining Kit (E607318; Sangon Biotech, Shanghai, China).

At the end of the study, the whole-body perfusion and fixation was performed once the animal is under general anesthesia. Then the livers and intestines were isolated and transferred into 30% sucrose for dehydration. Then the completely dehydrated tissues were embedded in O.C.T. compound. Then freeze and store the embedded tissues at -80°C freezer until sectioning. The frozen tissue blocks were sectioned at 10-μm thickness using a cryostat (Lecia CM 1950), then the sections were stained for histological investigation using Hematoxylin-eosin (H&E) staining kit (Sangon Biotech, E607318-0200) and Oil Red O staining kit (Sangon Biotech, E607319-0010), following the manufacturer’s instructions. H&E image of swiss-rolled intestine was divided into 4 quadrants and 10 intact villi from each quadrant were randomly selected for measurement. Villus length was then determined as the distance from the distal edge of the crypt base to the villus apex.