Panoramic scan slide scanner

Formalin-fixed tissues were cut into 3-μm sections by using a Leica RM2255 microtome (Leica Biosystems, Concord, ON, Canada). These sections were first stained with hematoxylin and eosin (H&E) and then scanned with a 3DHISTECH Panoramic SCAN slide scanner. The scanned image was further analyzed using Panoramic Viewer software. The placental composition was analyzed using ImageJ at 1.5 × magnification. A major product of DNA oxidation is 8-hydroxy-2-deoxyguanosine (8-OHdG), which is often used as a biomarker for oxidative stress (24 (link)). The oxidative stresses of both the placenta and fetal liver were determined using 8-OhdG (25 (link)). The tissue sections were transferred to polylysine-coated slides and incubated with primary anti-8-OhdG antibody (Santa Cruz Biotechnology, CA, USA) for 60 min at room temperature. After rinsing was conducted, the sections were incubated with secondary antibody for 30 min at room temperature and thereafter incubated with Avidin and biotinylated horseradish peroxidase H. The horseradish peroxidase converted the diaminobenzidine tetrahydrochloride substrate into an insoluble dark brown precipitate. To investigate the effects of a maternal HF diet on the fatty liver and the mechanism by which this developmental priming is mediated, fetal livers were also indicated for study.

Placental tissue was fixed in 4% paraformaldehyde and dehydrated through a gradient of ethanol and embedded. The tissues were sectioned (5 μm) on a tissue microtome. Subsequently, they were stained with hematoxylin and eosin (HE). Scans were carried out using a 3DHISTECH Panoramic SCAN slide scanner, and the images were analyzed using Panorama Viewer software (version 1.0.7). Placental composition and the area of blood sinusoids were assessed using ImageJ (version 1.48).

All skeletal muscles were taken out from −80°C and quickly transferred to the precooled constant cold box slicer (Leica, CM1900). The skeletal muscles were cryoprotected, and consecutive 10 μm-thick cross-sections were made. Importantly, during amelioration, the skeletal muscle tissue was kept in a frozen state, otherwise, it was extremely easy to form ice crystals. Continuous sections were stained with hematoxylin and eosin (H&E) to identify the general morphology and distribution of the muscle spindles. The following H&E routine steps were carried out: Frozen sections were recovered to room temperature and stained in hematoxylin for 3 min, color-separated with 1% hydrochloric acid and alcohol for 30 s, and treated with water for blue color return for 12 min. Then, the sample was stained in eosin for 1 min, dehydrated with gradient alcohol, and subjected to xylene clearance and subsequent neutral resin sealing. Finally, slides were digitally scanned using a Panoramic SCAN slide scanner (3D Histech, Hungary) with a 20 × or 40 × objective.