Ax10 microscope

Kidney tissues were collected and fixed in 4% paraformaldehyde for 24 h. After gradient dehydration, the kidney tissues were embedded in paraffin and finally cut into 3-μm sections. The HE staining was performed with a Hematoxylin and Eosin Staining Kit (Beyotime, Shanghai, China). Referring to the evaluation criteria from previous literature, we scored the pathological conditions of the glomerulus and interstitial cells by selecting different visual fields. Morphometric analyses of the glomerulus and renal tubules were determined using ImageJ. For immunostaining, the sections were incubated with anti-Fhl2 antibody (ab202584, Abcam, Cambridge, UK), anti-Lama2 antibody (ab11576, Abcam, Cambridge, UK), anti-DPP4 antibody (GB114937, Servicebio, Wuhan, China), anti-Fn1 antibody (GB114491, Servicebio, Wuhan, China) and pAKT (4060S, Cell Signaling Technology, Boston, MA, USA). After incubation with secondary antibodies for 2 h, the sections were sealed with antifade mounting medium with DAPI (041221210930, Beyotime, Shanghai, China). Images were acquired by a Zeiss AX10 microscope (Carl Zeiss, Weimar, Germany).

Ovaries were collected from OT-transplanted mice at 3 and 7 days. To explore the molecular mechanism of resveratrol, we tested relevant markers on the grafted ovary through immunohistochemical examination to validate the selected pathways. Sections were incubated for 1 h with primary antibodies against MDA (StressMarp, SMC-515, 1:800), SOD2 (Huabio, ET1701–54, 1:50), NF-κb (Huabio, R1309–9, 1:50), IL-6 (Huabio, R1412–2, 1:50), and SIRT1 (Huabio, ER1308–11, 1:50) at RT in a humid chamber, washed, and treated with EnVision goat anti-mouse IgG (Fuzhou Maixin, KIT-5006). A peroxidase substrate kit (SGK347011, Shanghai Gene) was used as a chromogen, and hematoxylin was used as a counterstain. Finally, six tissue sections from each group were obtained for analysis. The slides were examined under an inverted Zeiss AX10 microscope (Carl Zeiss, Germany). Brown coloring of the cytoplasm/nucleus of stromal cells, granulosa cells, or oocytes was defined as positive staining (any other coloring was considered negative staining).

For microscopy sample preparation, overnight cultures were diluted to OD₆₀₀ ~0.05 in fresh LB. Strains were induced with 1 mM IPTG and cultured at 37 °C with shaking. Cells were washed three times with PBS (pH 7.4) before observation, and examined using a ZEISS AX10 microscope (Carl Zeiss AG, Oberkochen, Germany) at different time points.