2.3. Homogenization of whole brain and supernatant
The brains of developing rats were collected from the sacrificed
pups at different ages i.e., 14th, 21st and 30th day. The
collected brains were homogenized 1:40 (w/v) in 0.1 M phosphate
buffer, pH 7.4, containing 1 mM EDTA and were centrifuged at
8000–10,000 rpm for 10–15 min. After centrifugation the supernatant
was collected and mixed with ice cold isopropanol and was
centrifuged to precipitate the proteins. The collected supernatant
was mixed with ice cold hexane and centrifuged again to extract all
nonpolar compounds into hexane and stored at −20 ◦C for further
analysis.