Confocal stacks of fixed brains were made using Zeiss LSM 510 or Leica SP6 confocal microscopes.
Frozen brains were embedded in Tissue Freezing Medium (Leica Instruments, Germany) at −20°C, and 6 8 µm-thick sagittal brain sections were made using the vibratome Microm HM500 (MICROM International, Germany).
Brain mapping figures were made using Caret (Van Essen, Dickson, Harwell, Hanlon, Anderson & Drury, 2001).
Brain and muscle lysates were made using PreCellys24 homogeniser (PeqLab) with lysis buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 1 mM NaF, 0.5 mM PMSF, 1 mM DTT, 10 mM glycerol phosphate and Complete Protease Inhibitors (Roche)) and then centrifuged for 10 minutes at 4000× g.
Brain sections (50 µm) were made using a cryostat (Leica) and mounted on glass slides in glycerol/PBS mix.
Coronal brain sections of 400 µm were made using a vibratome and the section(s) containing the SCN were selected based on surrounding anatomical landmarks when viewed with a dissecting microscope.
Following fixation the brain was embedded in optimal cutting compound (OCT) and 8μm coronal sections were made using a cryostat.
Recordings of brain activity can be made using electrodes, either glued to the scalp as in EEG studies, or implanted inside the brains of animals for extracellular recordings, which can detect action potentials generated by individual neurons.
A computerized Fourier analysis of the EEG waves will be made, using the Brain Wave Analyzer Software.
Counting of BrdU+ cells in GCL was made using 2 3 brain sections per mouse with each group of seven mice.
