Ba410t

The apoptosis of ARPE-19 cells was evaluated by the TUNEL apoptosis detection kit (40306ES50, YEASEN, China) [25 (link)]. Briefly, after the cell slides were fixed with 4% paraformaldehyde for 0.5 h, 100 μL proteinase K solution was added dropwise, and the reaction was performed at 37°C for 20 min. Then, the slides were added 100 μL of 1 × equilibration buffer and incubated at 25°C for 10 min. Finally, the slide was added 50 μL TdT buffer and incubated at 37°C for 1 h without light. The cell nucleus was stained with DAPI at 37°C for 10 min. Images were viewed and collected by a fluorescence microscope (BA410T, Motic).

We followed the instructions of the TUNEL kit (40306ES50, Shanghai Yisheng Bio, China). After the two groups of placental tissues were sliced, they were washed with PBS (pH = 7.2–7.6, Abiowell, China) for 3 times, and equilibration buffer was added and placed at room temperature for 10–30 min. Subsequently, 50 μL of TdT enzyme incubation buffer was added to the sectioned tissues and incubated at 37°C in the dark for 30 min. The nuclei were stained with DAPI working solution (Abiowell, China) for 10 min at the same temperature and rinsed with PBS. Buffered glycerol (Abiowell, China) was used to seal slices, and slices were observed under the ×400 field of view of the fluorescence microscope (BA410 T, Motic, China). If the nucleus was stained brown, it was a positive apoptotic cell.

Suspended cells were evenly distributed by pipetting, and a small amount of the suspension was then smeared on the slide and left to dry. Triton X-100 (0.3%) was used for 30 min at 37 °C for permeabilization. Then, 5% BSA was used for blocking at 37 °C for 60 min. A suitable dilution of the anti-LC3 primary antibody (14600-1-AP, 1:100, Proteintech) was added dropwise onto the slide for overnight incubation at 4℃. Then, the slide was incubated with 50–100 μL goat anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (A-11008, 1:200, Thermo Fisher) at 37 °C for 90 min. DAPI working solution was added for nuclear staining at 37 °C for 10 min. The slides were sealed with buffered glycerin and observed under a fluorescence microscope (BA410T, MOTIC).

To detect cell apoptosis, terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) assay staining was performed following manufacturers’ instructions (21 (link)). Embedded frozen liver tissue sections were prepared and stained with TUNEL reagents after sequential deparaffinization followed by an apoptosis in situ detection kit (Yeasen Biotechnology, Shanghai, China). 4′,6-diamidino-2-phenylindole (DAPI) staining was used to visualize the nuclei. TUNEL-positive cells labeled with fluorescein isothiocyanate were imaged via fluorescence microscopy (BA410T, Motic). The frequency of apoptotic cells in the liver section was semi-quantified by determining the percentage of TUNEL-positive cells in three microscopic fields per specimen.

Liver tissues were removed, fixed in 4% paraformaldehyde, then deparaffinized and processed through a series of increasing concentrations of ethanol for dehydration. These samples were subsequently paraffin-embedded and sectioned. The sections were then stained with hematoxylin and eosin for general histological assessment or the immunohistochemical analysis of anti-MIF primary antibody (20 (link)). Slides were subjected to 20 min of autoclave heating (0.01 Mcitrate buffer, pH 6.0) for antigen retrieval. After blockage of the endogenous activity of peroxidase by incubation with 1% periodic acid for 10 min, sections were then incubated with anti-MIF antibody (ab187064, Abcam) overnight at 4°C, followed by 30 min of incubation with secondary antibodies. Slides were finally visualized by diaminobenzidine (DAB) Chromogen for 5 min. After hematoxylin staining of the nucleus, all pieces were sealed with neutral gum and evaluated using a microscope (BA410T, Motic). The positive criterion was MIF showing fine, granular dark-brown depositions. Lipid accumulation was quantified by the area of lipid droplets (LDs) in liver tissues via Oil Red O (Wellbio, Changsha, China) staining. The staining images were analyzed by using ImageJ software.

The cell climbing piece was fixed with 4% paraformaldehyde for 30 minutes. Proteinase K working fluid was prepared. 100 μL 1 × equilibrium buffer was added to each sample and incubated at room temperature for 10–30 min. 50 μL TdT incubation buffer was added to the cell climbing piece. The DAPI (Wellbio, China) working fluid was dyed at 37°C for 10 min. The cell climbing piece was washed with PBS 5 min 3 times. The samples were stored in the dark and observed under a fluorescence microscope (BA410T, Motic, China).