Nonfat dry milk

In general, endogenous peroxidases were blocked with 3% H₂O₂ in methanol for 30 min followed by antigen retrieval. Blocking of non-specific binding was attained by an hour incubation with a block buffer containing 2% bovine serum albumin (Sigma-Aldrich, Belgium), 1% non-fat dry milk (Nestlé, Anderlecht, Belgium) and 0.1% Tween80, unless mentioned differently. Subsequently, the primary and secondary antibody were incubated and morphology was visualized by counterstaining with hematoxylin. Note that the slides were washed with Tris buffered saline between each step. Table 1 displays the specifics of each assay.

For tissue punches of hippocampus, hemisectioned brains were embedded rostral side down in OCT media and sectioned on a Leica (CM3050) cryostat at −25°C, and a 500–1000 μm 1 mm diameter punch from Interaural 1.10 to 2.10 mm (according to Paxinos & Franklin Mouse Brain Atlas, 2nd ed.) was taken for each MEST and Sham mouse from ZT1 and ZT15. Individual hippocampal punches were homogenized in 100 μl Tris-HCl buffer with Protease Inhibitor Cocktail (Sigma P2714) for 45 s and centrifuged at 14,000 rpm for 1 min, and 5 μl lysates were loaded with 5 μl 2X loading buffer and separated on 10% Tric-HCl 0.75 mm gels and transferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% nonfat dry milk (Nestlé) and blotted with BMAL (Arntl) antibody (1:1000; Bethyl Laboratories) and beta-actin antibody (1:1000; Cell Signaling). Goat anti-Rabbit IR800 (1:5000; LI-COR) secondary antibody was used to visualize on Odyssey scanner (LI-COR). Intensities of protein were determined using ImageJ software (NIH).