Abc elite

Paraffin embedded testicular sections were dewaxed and re-hydrated. Sections were immersed in solution of methanol:hydrogen peroxide (1:10) to block endogenous peroxidase activity. After antigen retrieving with boiling PBS, the sections were blocked with 10% goat serum at RT for 1 hour. And then, the primary antibodies were added on the sections and stored at 4℃ overnight. The primary antibodies we used in this study were including Ki67 (Abcam, USA), p16 (Santa Cruz Biotechnology, USA), p-p53 (Cell Signaling Technology,USA), 8-OHdG (Novus, USA) and γ.H2AX (Cell Signaling Technology, USA). The slides were rinsed in PBS and incubated with the biotin-conjugated secondary antibodies including goat anti-rabbit or goat anti-mouse (KPL, USA) at RT for 1hour. After being washed with PBS, the sections were incubated with Elite ABC (Vector, USA) at RT for 1 hour. And then, the sections were visualized by 3,3-diaminobenzidine (DAB) (Vector, USA) and counter-stained with Hematoxylin. The micrographs were taken.

The human and mouse LF samples collected were fixed in a 4% paraformaldehyde solution overnight at 4 °C; decalcified in an EDTA (ethylene diamine tetra-acetic acid)-glycerol solution for 14–21 days at 4 °C; successively dehydrated, hyalinised, and embedded in paraffin; and then sliced into 5-μm-thick sections for histological staining. The dewaxed and rehydrated sections were subjected to haematoxylin-eosin (H&E; Jiancheng, Nanjing, China) or EVG staining (Baso, Zhuhai, China), in accordance with the manufacturer's instructions. For immunohistochemical (IHC) staining, the sections were incubated overnight at 4 °C using the primary antibodies listed in Table S2. The sections were washed with PBS three times and successively incubated with biotin-conjugated secondary antibodies for an hour, Elite ABC (Vector, USA) for an hour, and diaminobenzidine (Vector) at room temperature. After counterstaining with haematoxylin, the sections were observed under a microscope.
All the images were photographed using the Leica LAS-X software, under the Leica DMi8 microscope. The IHC staining images were analysed with Image Pro Plus. In accordance with the previously described method [32 (link)], quantitative analyses of the LF areas and the ratio of the elastic fibres to the collagen fibres in mice or human LF samples were performed using the ImageJ software (NIH).