Adult female mice at proestrus or diestrus stage were sacrificed by exposure to increasing concentration of CO2 at six different time points (ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20; n = 5 per experimental point). After intracardiac blood puncture, mice were intracardially perfused with 10 mL phosphate buffer saline 0.1 M (PBS, pH 7.4) followed by 20 mL of periodate-lysine-paraformaldehyde fixative (formaldehyde 4%, NaIO4 10 mM and lysine 75 mM in 100 mM phosphate buffer, pH 7.3). The brains were collected, post-fixated in periodate-lysine-paraformaldehyde for 12 hours, washed out with PBS, dehydrated and embedded in polyethylene glycol as previously described [44].

Twelve series of 12 μm-thick coronal brain sections were cut using a microtome throughout the DMH as presented in the Paxinos mouse brain atlas. For each mouse, one section in every twelve (i.e. 1 section every 144 µm giving 6–7 DMH-containing brain sections) was rehydrated and mounted on a SuperFrost Plus (Menzel-Glaser, Braunschweig, Germany) slide. For each immunolabeling, DMH-containing slices of all mice of different time points and estrous stages were processed at the same time in order to limit variations in the labeling background.

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