Journal: Biology of reproduction

Article Title: NQO2 inhibition relieves reactive oxygen species effects on mouse oocyte meiotic maturation and embryo development.

doi: 10.1093/biolre/iox098

Figure Lengend Snippet: Figure 2. NQO2 relationship with microtubules and chromosomes during oocyte meiotic progression. Oocytes were collected at 2, 4, 8, 10, 12, and 17 h of matu- ration culture, corresponding to GVBD, pro-MI, MI, AI, TI, and MII stages, respectively. NQO2 was visualized in red, acetylated-tubulin (ace-tubulin) or centromere was visualized in green, and DNA was visualized in blue. (A) NQO2 was presented as filamentous aggregates and specially co-localized with microtubules. Bar = 20 μm. (B) Immunostaining analysis revealed NQO2 accumulation across chromosomes. Scale bar = 10 μm. (C) Negative control of NQO2 immunostaining. No fluorescence signal of NQO2 was detected in the spindle area of oocytes or across chromosome spreading samples after immunostaining without primary antibody to NQO2. a–d, scale bar = 20 μm; e–h, scale bar = 10 μm. (D) NQO2 distribution was tightly associated with microtubule integrity and normal dynamics in oocytes. Oocytes at MI stage were treated with spindle-disturbing agents, nocodazole and taxol, and then fixed for immunostaining with ace-tubulin and NQO2. Scale bar = 20 μm.

Article Snippet: After thoroughly washed in phosphate-buffered saline (PBS) containing 0.02% Triton X-100 (PBST), the oocytes were blocked in PBS containing 10% normal goat serum and 1% BSA at 4◦C overnight, and then incubated in diluted primary antibodies, including rabbit polyclonal anti-NQO2 (1:250; GTX105899, GeneTex), mouse monoclonal anti-Lamin A (1:250; ab8980, Abcam), mouse monoclonal anti-acetylated tubulin (1:10 000; T7451, Sigma), and human centromere auto serum (CREST) (1:500; 90C-CS1058, Fitzgerald) at 4◦C overnight.

Techniques: Immunostaining, Negative Control