Bx50 fluorescence microscope

The ovaries and parametrial fat samples were collected from control, fostered PNA, and PNA mice at 16 weeks of age. The ovaries and adipose tissue were fixed, embedded in paraffin, and 5-μm thick sections were prepared. These sections were stained with hematoxylin and eosin. The ovarian sections were examined by two investigators using an Olympus BX50 Fluorescence Microscope (Olympus, Tokyo, Japan). The number of atretic antral follicles were counted across the entire ovary, as previously described (Kusamoto et al., 2021 (link)). Atretic follicles were recognized by the presence of an attenuated granulosa cell layer, shrinkage, and degenerate oocyte nuclei (Osman, 1985 (link)). For the adipose tissue samples, two representative micrographs were obtained per sample at ×40 magnification using a light microscope (Olympus BX50 Fluorescence Microscope). The sizes of the adipocytes were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States), as previously described (Benrick et al., 2017 (link); Kusamoto et al., 2021 (link)).

To detect the nuclear translocations of NF-κB and Nrf2, RAW 264.7 cells were cultured directly on glass cover slips in 6-well plates and treated with 300 μg/mL SC-E3 in the presence or absence of LPS. Briefly, cells were fixed with methanol for 10 min, permeabilized in PBS containing 1% Triton X-100 for 10 min, incubated with NF-κB p65 or Nrf2 antibody (1 : 200) in PBS overnight at 4°C, and labelled with fluorescein isothiocyanate- (FITC-) conjugated goat anti-rabbit IgG (1 : 1000, Invitrogen) for 1 h and DAPI (Sigma-Aldrich) for 5 min. After mounting coverslips on glass slides using ProLong® Gold Antifade Reagent (Thermo Scientific), fluorescence images were captured using an Olympus BX50 fluorescence microscope (Olympus Optical, Tokyo, Japan).

Following the completion of the experiments, all the rats were sacrificed by exsanguination under isoflurane anesthesia (ABBOTT, Abbott Laboratories Ltd., Queenborough, Kent, England). A laminectomy was performed at the lower level of the twelfth thoracic vertebra, and the L5–S3 segment of the spinal cord was removed and embedded in an optimal cutting temperature compound (Sakura Finetec Inc., USA) for immunohistochemistry.
Spinal cord sections (5μm) were fixed by soaking in ice-cold acetone/methanol (1:1) for 5 min. After three washes in ice-cold phosphate-buffered saline (PBS), sections were categorized by incubation overnight at 4°C with the FITC-labeled mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody for astrocytes (Molecular Probes, Oregon, USA), the FITC-labeled mouse monoclonal anti-rat CD11b/c antibody for microglia (OX42; Serotec, Oxford, UK), and the FITC-labeled mouse monoclonal anti-neuronal nuclei for neurons (Chemicon, Temecula, CA) diluted in 1% normal goat serum in PBS. After three PBS rinses, 400x magnification images were taken using an Olympus BX 50 fluorescence microscope (Olympus Optical, Tokyo, Japan) and a Delta Vision disconsolation microscopic system operated using SPOT software (Diagnostic Instruments Inc., USA). For each rat, three images were produced for the three cells astrocytes, microglia, and neurons.