Sample preparation for AFM

After thawing, cryopreserved ovarian biopsies were immediately embedded in optimal cutting temperature compound (OCT Tissue-Tek, VWR International, Edmonton, Canada) and snap-frozen in liquid nitrogen, without prior labeling, fixation, or dehydration. Fifty-µm-thick ovarian cortex cryosections were obtained using a cryostat (Microm HM 560, Walldorf, Germany) and mounted on Superfrost Plus glass slides (Menzel-Glaser, Germany). The first 200 µm from the surface was cut away to go beyond the mesothelial layer, and tissue slides 300–500 µm in depth within the ovarian cortex were selected for AFM analyses. On the day of AFM analyses, tissue sections were incubated at room temperature for 5 min to thaw the OCT compound and reinforce tissue adhesion to slides. The slides were then rinsed in pure water several times to remove excess OCT and incubated in PBS at room temperature for 20 min to equilibrate ion charges while conducting AFM calibration steps, as detailed below. Finally, the samples were mounted for AFM and covered with fresh PBS to conduct biomechanical characterization.