Sc 133080

Sliced sections were air dried and incubated in frozen methanol (2 min) and in 4% paraformalaldehyde (4 min). After three washes in phosphate buffer saline (PBS) containing Tween 20 (PBS/T) (Sigma-Aldrich), the sections were incubated for 60 min in a blocking solution (normal goat serum, in PBS) and then overnight at 4 °C with the primary antibodies against neuropeptide Y (NPY) (mouse monoclonal anti-NPY antiserum (1:500), product code: sc-133080, Santa Cruz Biotechnology, Inc. Heidelberg Germany), brain-derived neurotrophic factor (BDNF) (rabbit polyclonal anti-BDNF antiserum (1:300), product code: sc-ANT-010, Alomone Labs, Jerusalem Israel), glial fibrillary acidic protein (GFAP) (mouse polyclonal anti-GFAP antiserum (1:200), product code: G3893, Sigma-Saint Louis, MO, USA) and tau protein (rabbit polyclonal anti-tau antiserum (1:1000), product code: Ab-64193, Abcam Israel). After three washes in PBS/T, sections were incubated for 2 h in DyLight-488 labeled goat anti-rabbit IgG (BNDF: 1/1250; tau: 1/1750) or in Dylight-594 goat anti-mouse IgG (NPY: 1/500; GFAP: 1/500; KPL, MD, USA) in PBS containing 2% normal goat or horse serum.

PVN tissues were dissected out and homogenized in an ice-cold lysing buffer. Following determining the concentration of protein for each lysate, the proteins were loaded and separated by running in a SDS-PAGE gel, followed by electrophoretically transferring the protein to the nitrocellulose membranes (Millipore, Billerica, MA, United States). After being blocked overnight at 4°C using blocking buffer, the membrane was incubated with the primary antibodies at 4°C overnight, including mouse monoclonal anti-NPY (1 : 1000 dilution; #sc-133080, Santa Cruz Biotechnology), mouse monoclonal anti-NPY1-R (1 : 1000 dilution; #sc-393192, Santa Cruz Biotechnology), or NPY5-R (1 : 1000 dilution; #sc-137167, Santa Cruz Biotechnology). Then the membrane was incubated with conjugated secondary antibody at room temperature for 1 h and developed in the electrochemiluminescence (ECL). The images were collected from the film and identified by the Quantity One Software (Bio-Rad, Hercules, CA). The target protein levels were expressed as relative values against the β-actin level on the same gel.