Twenty-four mice (male, 8 weeks old) feeding on a standard chow diet were equally divided into eight groups (NC-0h, NC-1h, NC-2h, NC-3h; D3-0h, D3-1h, D3-2h, D3-3h). For gene expression assays, the hypothalamus was harvested 0–3 hours following D3 administration (NC: saline), and the expression levels of c-Fos, AgRp and Pomc were quantified by quantitative RT-PCR.
The same experiment was carried out for the immunofluorescence. c-Fos staining was performed as described in a previous study.66 (link) Briefly, mice were anaesthetised with a lethal dose of pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, placed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 1 week. Brains were cut into 25 mm sections. The sections were treated as described above and incubated overnight at room temperature in mouse anti-FOS (1:500; ab208942; Abcam) or rabbit anti-POMC (1:100; ab254257; Abcam). Detection and labelling were performed using secondary antibodies conjugated to Alexa Fluor-594 donkey anti-mouse IgG (H+L) (1/500, ab150108, Abcam) or Alexa Fluor-488 donkey anti-rabbit IgG (H+L) (1/500, ab150073, Abcam), and imaging was performed as described above. Images were pseudocoloured using Photoshop software (Adobe) or ImageJ (NIH).
The same experiment was carried out for the immunofluorescence. c-Fos staining was performed as described in a previous study.66 (link) Briefly, mice were anaesthetised with a lethal dose of pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, placed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 1 week. Brains were cut into 25 mm sections. The sections were treated as described above and incubated overnight at room temperature in mouse anti-FOS (1:500; ab208942; Abcam) or rabbit anti-POMC (1:100; ab254257; Abcam). Detection and labelling were performed using secondary antibodies conjugated to Alexa Fluor-594 donkey anti-mouse IgG (H+L) (1/500, ab150108, Abcam) or Alexa Fluor-488 donkey anti-rabbit IgG (H+L) (1/500, ab150073, Abcam), and imaging was performed as described above. Images were pseudocoloured using Photoshop software (Adobe) or ImageJ (NIH).
