Bs 0295g af488

Ovarian tissues were fixed with 4% paraformaldehyde for 24 h at room temperature. Tissue sections underwent microwave-assisted antigen retrieval at 92–96 °C for 15 min, followed by blocking with 5% BSA at 37 °C for 1 h. The sections were then incubated with FSHR antibody (1:200; AF5242; Affinity, USA) overnight at 4 °C, followed by a 1 h incubation at 37 °C with Goat Anti-Rabbit IgG H&L/AF488 secondary antibody (1:200; bs-0295G-AF488; Bioss, China). Finally, the sections were counterstained with DAPI and visualized under a fluorescence microscope (BX53; Olympus, Tokyo, Japan).

Mice were perfused with 4% paraformaldehyde at 4 °C after sacrifice. After complete brain dissection, gradient sucrose solution was used for dehydration. The mouse brain was embedded in O.C.T. compound (Sakura, 4583) and coronally sliced at −25 °C (20 μm) in a freezing microtome. The slices were washed with PBS, and slices of the substantia nigra were selected by soft brushing. The slides were blocked with goat serum (Solarbio, SL038) for 30 min, then placed in 1% BSA and anti-TH antibody (ProteinTech, 25859-1-AP) at 1:500 and incubated overnight at 4 °C. The fluorescent secondary antibody anti-488 (Bioss, bs-0295G-AF488) was diluted at 1:1000 in 1% BSA and incubated with the slides at room temperature for 1 h (eGFP-mCherry-LC3 mice were stained with DAPI for 5 min after brain sectioning). After washing with PBS three times, the slides were sealed with glycerin. All images were obtained by fluorescence microscopy (Olympus, BX53).

The positive expressions of Aβ, Tau, p-Tau, and YTHDF1 in hippocampal and cortical tissues were detected by immunofluorescence. Briefly, frozen slices of tissues were made after paraformaldehyde perfusion. The slices were baked in an oven at 60°C for 8 h and then repaired twice with sodium citrate repair solution for 5 min each. After washing with PBS and shaking dry, 5% BSA was closed at 37°C for 1.5 h. Afterward, the following primary antibodies were added: rabbit antiAβ1-42 (Ab201060, Abcam), rabbit anti-Tau (AB37439-100, Multi-Science), rabbit antiphospho S199 (Ab 81268, Abcam), rabbit anti- YTHDF1 (17479-1-AP, Proteintech), for 2 h at 37°C. Then, the sections were incubated overnight at 4°C. The next day, the following secondary antibodies were added after washing with PBS: goat antirabbit IgG/Alexa Fluor (BS-0295G-AF488, Bioss), and incubated at 37°C for 1.5 h. DAPI staining solution (C1005, Beyotime) was added dropwise, and PBS was used to wash off DAPI and seal the slices after 5 min. Finally, observation was performed with a fluorescence microscope (TSC SP8, Leica).