Rm2016

Sections of brain tissue (five slices per sample) were collected, fixed, dehydrated, and transparently rendered in ethanol and xylene solution. The wax-soaked tissue is embedded in the embedding machine. Place the trimmed wax block cool at −20 °C freezing table, and slice the modified tissue chip wax block on the paraffin slicer(Leica RM2016 Shanghai, China), the slice thickness is 4 μm. Stain sections with Hematoxylin (HE dye solution set, Servicebio, Wuhan, China) solution for 3–5 min, and rinse with tap water. Then treat the section with hematoxylin differentiation solution, and rinse with tap water. Treat the section with Hematoxylin Scott Tap Bluing, and rinse with tap water. A total of 85% ethanol (SUPELCO, 1009832500) for 5 min; 95% ethanol for 5 min; finally stain sections with eosin dye for 5 min. Photomicrographs were captured with a light microscope (Nikon Eclipse E100, Tokyo, Japan). The nucleus was blue-purple, and the cytoplasm was red.
To calculate the number of abnormal neurons, three brain slices were randomly selected, and five nonoverlapping high-power fields (×400) in the hippocampal CA1 sector were randomly selected in each slice. Then, the number of abnormal neurons in the regions was calculated. By calculating the average number of abnormal neurons, statistical analysis was acquired for each group of samples.

Transfected cells were fixed with 2.5% glutaraldehyde (G105907, Aladdin, China) at 4°C overnight, followed by 2% osmium tetroxide (18466, TED PELLA, USA). After the cells had been stained with 1% uranyl acetate (CD106833, Codow, China), they were dehydrated with a gradient concentration of ethanol (100092683, Sinopharm, China) and acetone (10000418, Sinopharm), embedded in epoxy resin (XW00000020, Sinopharm), and sectioned using an ultramicrotome (RM2016, Leica, China). Autophagosomes were observed using TEM (Hitachi, Tokyo, Japan).

The placental tissues preserved in 4% formalin were obtained, dehydrated in gradient alcohol (75, 80, 90, 95 and 100%) twice for 1.5 h each series, and cleared with xylene twice for 8 min each time. Subsequently, the tissues were embedded in paraffin and sliced into 5-7-µm-thick slices using a microtome (RM2016; Leica, Shanghai, China). The slices were then incubated at 55°C, deparaffinized in xylene and hydrated with gradient alcohol at concentrations of 100, 95, 85 and 75% (3 min each concentration). The tissues were then stained with hematoxylin for 8 min, washed with water for 2 min, differentiated with hydrochloric alcohol for 5 sec, and washed with water for 2 min. The tissues were treated with 0.25% ammonia for 1 min and washed with water for 2 min. Subsequently, the tissues were stained using eosin for 30 sec and washed with water for 2 min prior to dehydration twice with a gradient alcohol series (85, 95 and 100%; 2 min for each) and xylene clearing twice (5 min each). When the staining was completed, the sample slides were mounted using neutral balsam and sealed with clean cover slips. The histopathological changes in the placenta were observed under a microscope (XSP-2C; Bingyu Optical Instrument Co., Ltd., Shanghai, China).