Ab64264

The antibodies used in the present study were anti-SREBP (sc-8984, rabbit polyclonal, dilution 1:2000, Santa Cruz Biotechnology), anti-SCAP (bs-3862R, rabbit polyclonal, dilution 1:500, Bioss), anti-LIMP II (sc-25869, goat polyclonal, dilution 1:300, Santa Cruz Biotechnology), and anti-CD36 (sc-5523, goat polyclonal, dilution 1:800, Santa Cruz Biotechnology).
For immunohistochemical analysis, paraffin sections were deparaffinized and rehydrated through graded alcohols; antigen retrieval was performed in 10 mM citrate buffer pH 6.0, at 97°C for 5 min. The blockade of endogenous peroxidases was obtained by covering the slides with hydrogen peroxide Block (ab64264, abcam) for 15 min and the blockade of non-specific protein–protein interactions was achieved by incubating sections with Protein Block (ab64264, abcam) for 15 min. After pretreatment, the sections were incubated overnight at 4°C with the antibodies diluted in 1% Tris Buffer Solution. Subsequently, slides were incubated with biotinylated Goat anti-polyvalent secondary antibody (ab64264, abcam) for 10 min. The positive signals were visualized as brown precipitates utilizing 3-30-diaminobenzidine tetrahydrochloride (DAB, Sigma) solution. Hematoxylin was used for counterstaining.

Ovaries were fixed in cold 4% paraformaldehyde overnight, incubated sequentially in 10% and 20% sucrose in PBS overnight, embedded in OCT (optimal cutting temperature medium), and stored at −80 °C until cryosectioning. After high-temperature antigen retrieval with 0.01% sodium citrate buffer (pH 6.0), the frozen sections (10 μm) were blocked with 10% normal donkey serum for 30 min and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used are presented in SI Appendix, Table S2. For immunofluorescence, the sections were washed with wash buffer and incubated with the appropriate Alexa-Fluor–conjugated secondary antibodies (1:200; Invitrogen) at room temperature for 2 h. After staining with DAPI, samples were analyzed using confocal microscopy (Leica SP5). For immunohistochemistry, the slides were incubated with avidin-conjugated secondary antibodies (ab64264; Abcam) before being exposed to diaminobenzidine (DAB) (ab64264, Abcam) for 1 min and then counterstained with hematoxylin.

Histological sections were immunohistochemically stained to visualise the MRP1 expression and to study the proliferative state of the tumours. Microtome sectioned tissue specimens of 5 µm were dehydrated at 50 °C overnight, followed by deparaffinisation and rehydration. Rehydrated slides were first incubated in sodium citrate buffer pH 6 at 95 °C for 30 min for antigen retrieval and cooled down to room temperature. For ki67 staining, slides were additionally incubated with 0.1% Triton X-100 in PBS for 10 min to permeablise the nuclear envelope. Slides were then incubated in 1% H₂O₂ for 15 min to quench endogenous peroxidase activity, followed by serum blocking for 1 h. Goat anti-mouse IgG Fab (Abcam, ab6668) was added for 1 h to block endogenous IgG. After washing in PBST, slides were incubated with primary against ki67 (Abcam, ab8191) or primary against MRP1 (Santa Cruz, sc-18835) overnight in a moisture chamber at 4 °C. Slides were then incubated with biotinylated mouse-rabbit polyvalent secondary (Abcam, ab64264), followed by streptavidin peroxidase (Abcam, ab64264) incubation. After washing, slides were developed using 3,3′-diaminobenzidine (DAB) substrate for 15 min then counterstained with Mayer’s haematoxylin QS (Vector, H-3404), and eventually mounted in coverslips with DPX.