Mice were anesthetized using pentobarbital (Beuthanasia, Intervet Inc ., perfused with normal saline and brains were collected in 3 ml of HBSS (Gendepot, Barker, TX) containing 20% FBS, and homogenized using the plunger portion of a 5 ml syringe in six-well flat bottom plates.
Brains were collected in both the morning and the afternoon to increase chances of including circadian-controlled transcripts.
Briefly, fresh brains were collected in which six 300-μm coronal slices were prepared from each animal using a microtome (Leica VT1200, Leica Microsystems AB, Sweden).
Pregnant females were killed by cervical dislocation at appropriate time points and brains were collected in ice-cold PBS as previously described.
Briefly, 100 150 μm free-floating vibratome sections of brains were collected in PBS, blocked with 5% normal serum in PBS with 0.1% Triton X-100 and incubated with primary antibodies in blocking solution overnight at 4°C.
Brains were collected in the 17th light cycle (where the largest behavioral difference between WT and Per3 KO mice had been observed; ref. 13) and stored at −80°C in RNAlater (Qiagen).
Brains were collected in the Section on Neuropathology of the Clinical Brain Disorders Branch (CBDB) of the NIMH and the Stanley Foundation through the medical examiner offices following autopsy and through donations via funeral homes with consent from the next of kin.
The sections of mice brain were collected consecutively in PBS for immunofluorescence staining.
Anatomical images of each subject's brain were collected using an in-house multiecho 3D fast angle low shot (FLASH) sequence for mapping proton density, T1, and magnetization transfer, from which T1 weighted images were generated (voxel size, 1 × 1 × 1.5 mm) [ 27].
