Ab243698

The expression of MTR, BHMT, LC3B, and P62 in the ovaries was detected using IHC. The sections were baked at 65°C in an oven, dehydrated with alcohol, incubated with 3% H₂O₂, boiled in 10 mM citrate buffer (pH 6.0) for antigen retrieval, rinsed in Tris-buffered saline (TBS) three times, added to solution A (for biotin block), and washed three more times in TBS. Sections were incubated in goat serum for 1 h at room temperature, and were then incubated separately with anti-MTR (1:200, ab66039, Abcam, UK), anti-BHMT (1:200, ab243698, Abcam, UK), anti-LC3B (1:300, ab48394, Abcam, UK), and anti-P62 (1:200, F032602, Abways Technology) overnight at 4°C. Samples were then washed in PBS. Sections were incubated with a goat anti-rabbit secondary antibody at 37°C for 30 min, and the sections were then stained with 3,3'-diaminobenzidine and counterstained with hematoxylin at room temperature. The negative control sections were incubated in biotinylated goat antirabbit immunoglobulin G (1:200 dilution) and stained with the experimental sections. Densitometric quantification was performed using ImageJ software.

Lysates from ovarian tissue and mGCs were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with MTR antibody (1:2000, ab66039, Abcam, UK), BHMT antibody (1:200, ab243698, Abcam, UK), LC3B antibody (1:1000, ab48394, Abcam, UK), P62 antibody (1:1000, F032602, Abways Technology), Beclin-1 antibody (D40C5, 1:500, 3495s, Cell Signaling Technology, Inc.), mTOR (s2442) antibody (1:1000, BS3611, bioWORLD), and p-mTOR antibody (1:1000, BS4706, bioWORLD). In addition, samples were incubated with peroxidase-conjugated secondary antibody, and an enhanced chemiluminescence detection kit (Amersham Pharmacia Biosciences, Inc.) was used for visualization. Protein levels were quantified and normalized using glyceraldehyde 3-phosphate dehydrogenase (1:10,000, AB0037, Abways Technology) as internal control.