Nie a1 plus

hMOF-overexpressed OVCAR3 cells were seeded into plates containing glass coverslips and cultured for 24 h. The cell climbing slice was fixed with 4% paraformaldehyde for 2 h, permeabilized in 0.5% PBST (PBS containing 0.5% Triton X-100) for 10 min and blocked with 5% goat serum for 60 min. Then the cell slides were incubated with primary antibodies of hMOF (#ab200660, 1:1000, Abcam) and MDM2 (#66511-1-Ig, 1:500, Proteintech) at 4 °C overnight. After incubation with secondary antibodies (Alexa fluor 488 goat anti-rabbit, A0423, Beyotime, 1:1000; Alexa fluor 555 donkey anti-mouse, A0460, Beyotime, 1:1000) for 2 h at room temperature, the nuclei were counterstained with DAPI (1 μg/mL) for 5 min. The immunofluorescence images were captured by a Nikon NiE-A1 plus or Nikon C2+ confocal microscope, and analyzed using Nikon-elements AR software.

Tissues for immunofluorescence were fixed in 4% PFA solution for 24 h and then immersed in 30% sucrose for 24 h twice. Processed brain tissue was coronal sectioned at a thickness of 30 μm in four series (8–10 slices per series). Each series was incubated with primary antibodies against TH (1:500, Millipore AB152), DAT (1:100, Millipore MAB369), GFAP (1:300, DAKO Z0334), or Iba1 (1:500, Wako 019-19741), respectively, at 4 ℃ overnight, followed by secondary antibodies incubation at room temperature for 2 h. Images were obtained by confocal microscope (NiE-A1 plus, Nikon) or fluorescent microscope (E80i, Nikon) and processed by ImageJ (1.49c). The number of TH-positive cells was quantified within area of compact part of SN. The density of DAT signal was quantified by sampling 5 repeated 50 × 50 pixel areas within striatum. All quantification was single-blinded.