Deadend colorimetric tunel system

To visualize apoptotic cells, we used the DeadEndTM Colorimetric TUNEL System (Promega, Madison, WI) to determine the apoptotic cells according to the manufacturer’s instructions. The nuclei were counterstained with DAPI. Five retinal sections were randomly selected per each condition. After the length of each section was measured (Image-Pro Plus software), the TUNEL-positive cells were counted in the whole section length (between ora serrata to ora serrata). The number of apoptotic cells was expressed per 200 μm of retinal section57 (link).

A section of each tumor was fixed in 10% formalin for IHC determinations. Sections of 3–4 μm were cut from each sample by deparaffinizing and dehydrating using Hemo-De (Fisher Scientific, Pittsburgh, PA, USA), followed by an alcohol series and washing in phosphate-buffered saline (PBS), pH 7.0. The slides were then incubated in 3% H₂O₂ for 20 min at room temperature to block peroxidase, and antigens were retrieved by boiling the slides in a microwave oven in 50 mM citrate buffer, pH 6.4. After blocking with goat serum, the slides were incubated with PCNA, Ki67, and cleaved caspase-3 antibodies (Google Biological Science and Technology Co., Ltd., Wuhan, China). The slides were counterstained with hematoxylin. H&E stained sections were also made. Apoptosis was detected by the TUNEL assay using the DeadEnd^TM Colorimetric TUNEL System (Promega, Madison, WI, USA) per manufacturer’s protocol. Three random microscopic fields were captured for each tumor section at 200× magnification.

Mouse livers were fixed with 10% formalin and embedded in paraffin. Paraffin-embedded liver sections were stained with H&E for morphological analysis. Sirius red staining was performed using a Picrosirius Red stain kit according to the manufacturer’s instructions (Abcam, Cambridge, MA, UK). For the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, paraffin-embedded sections were stained using the DeadEnd^TM colorimetric TUNEL system according to the manufacturer’s instructions (Promega Corporation, Madison, WI, USA).
For Oil Red O staining, fresh mouse livers were embedded in OCT solution, and frozen liver sections were fixed in 10% formalin for 10 min and washed with distilled water. Air-dried sections were stained with Oil Red O solution for 15 min and washed with 60% isopropanol. Then, stained sections were washed with distilled water.

To analyze apoptosis after xenotransplantation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (DeadEndTM Colorimetric TUNEL System, Promega, Austria) was performed according to the manufacturer’s instructions, with 3,3-diaminobenzidine tetrahydrochloride (DAB) as chromogen. Mayer’s hemalaun solution (Merck Millipore, Germany) was used for counterstaining. Only sections where follicles were detected in the adjacent H&E-stained sections were analyzed. The TUNEL staining results were evaluated by three independent observers.
Follicles consisting of ≥49 % TUNEL positive granulosa cells as well as follicles containing a TUNEL positive oocyte were classified as atretic follicles [49 (link)]. Sections without transferase enzyme and human lymph node sections treated with DNAse served as negative and positive control, respectively.

TUNEL assay was conducted using the DeadEnd^TM colorimetric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s protocol [24 (link)]. The slides were viewed under bright-field inverted microscope (Nikon, Tokyo, Japan).

To confirm the apoptosis level of tumor in mice, paraffin tissue sections of tumors were processed with the DeadEnd^TM Colorimetric TUNEL System (Promega, Madison, WI) according to the manufacturer's protocol.

BPA (CAS no. 80-05-7) was from Sigma Chemical Company (Saint Louis, MO, USA). Rabbit anti-CYP11A1 (ab175408), 3β-HSD (ab65156), StAR (ab203193), Bcl-2 (ab59348), and Bax (ab53154) primary antibody and secondary antibody anti-rabbit were from Abcam (Cambridge, MA). The DeadEndTM Colorimetric TUNEL System was from Promega Corporation (Promega, Madison, WI, USA).