Ez c1 3.60 build 770 gold version

Brain sections (20 μm) were collected from plate 18 to plate 23 of the stereotaxic atlas (S2) to cover the whole PVN and arcuate area of the hypothalamus. Every fifth section was used for staining peptide. Brain slides were fixed with 4% paraformaldehyde for 20 min, and then washed with PBS twice. They were then fixed with methanol for 10 min and washed again with PBS. The brain sections were then blocked with 2.5% horse serum for 1 h and incubated with mouse anti-TH (1:500) antibody for 2 h or rabbit anti-CRH antibody (1:2000) overnight. For double staining, sections were incubated with rabbit anti β-EP (1:2000, Invitrogen) antibody and mouse anti-BrdU antibody (1:2000) overnight. The sections were then washed twice and incubated with Alexa anti-mouse IgG (1:500) and FITC anti-rabbit IgG (1:500) for 45 min. Stained slides were mounted with DAPI, and pictures were taken by a confocal microscope with a 20× objective lens (Nikon EZ-C1 3.60 build 770, Gold version). Cell numbers on each section were manually counted, and the sum of the total cell number from all sections of one brain was calculated.