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Young (3 months) and old (22 months) F344 rats were exercised once a day for 50 min at night onset over an 8-week period.
Horizontal and vertical activities were monitored for 10 min at night (complete darkness) in an automated Omnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH).
A light was placed inside this house and the door was opened for 30 min at night.
The waiting time was 25.0 min at night, and could be as much as 36.4 min during the day on the weekend.
The readings should be taken every 20 30 min during the day and every 30 60 min at night to avoid interfering with activity or sleep.
Blood pressure readings were obtained every 20 min during the day (7:00 a.m. 11 00 p.m). and every 30 min at night (11.00 p.m.–7:00 a.m .. Day- and nighttime periods were defined based on individual sleeping time.
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Nuclei were stained with 0.5 ml 1 µg ml−1 Hoechst (Molecular Probes H1399) for 10 min at 4 °C.
Hematoxylin and eosin staining was performed on 7-μm-thick cryosections fixed with 4% paraformaldehyde for 10 min at 4 °C.
Precipitates were rinsed three times with washing buffer (50 mM Tris/HCl, pH 7.4, 500 mM NaCl, 2 mM EDTA, 0.2% Igepal) for 10 min at 4 °C.
O3 application promoted the removal of more than 80% of pesticides when the roots were exposed for approximately 120 min at 5 and 10 mg L−1, respectively, in treatments with O3 as gas and dissolved in water.
Then, the homogenized material was resuspended in 100 μl extraction buffer (50 mM Tris HCl pH 7.5, 20% (v/v) glycerol, 1 mM EDTA pH 8.0, 10 mM β-mercaptoethanol) and centrifuged at 15,000g for 15 min at 4 °C.
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