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We biopsied 1-cm portions of tail from 4-week-old mice under ether anesthesia.
Tumor pieces were implanted subcutaneously uni-or bilaterally into the flanks of six to eight week old female mice under ether anaesthesia for a short period of time.
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At particular time points, blood (150 µl) was taken from the retro orbital vein of each mouse under ether anesthesia.
To induce delayed implantation, pregnant mice were ovariectomized under ether anesthesia at 08 30 09 00 h on day 4 of pregnancy.
The mice were killed under ether anaesthesia.
To obtain their brains, mice were killed under ether anesthesia.
At the end of the experiments, the mice were killed under ether anaesthesia and their tumours were examined as detailed below.
All the mice were killed under ether anaesthesia at 28 days after implantation for evaluation of the arising tumours' malignancy and autopsy; simultaneously we removed the subcutaneously growing tumours aseptically to assess whether the arising tumours had acquired malignant phenotype, and used them for establishing individual culture cell lines after mechanical disaggregation with scissors.
LM8 cells (1.05 × 106 cells/0.3 ml of PBS) were s.c. implanted in the backs of 4-week-old male BALB/cA Jcl- nu nude mice (Clea Japan, Inc., Tokyo, Japan) under ether anesthesia.
Cochleae from the temporal bones of 3-day-old Occ +/+ or Occ −/− mice under deep anesthesia with ether were dissected in PBS without calcium.
Mice were sacrificed by bleeding under ether anesthesia, following a method previously described [ 30].
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