Rabbit anti npy

Animals were perfused with ice-cold 4% PFA in PBS. Brains were removed, post-fixed overnight at 4 °C in 4% PFA, and rinsed 3 times in PBS. Prior to sectioning, brains were embedded in a BSA-gelatin mixture consisting of 0.4% gelatin, 23.3% BSA, 5.9% formalin, and 0.32% glutaraldehyde. Brains were then sectioned at 40–50 µm on a Leica vibratome. Sections were blocked and permeabilized in 10% normal donkey serum (NDS) with 0.4% Triton X-100 in PBS for 1–3 hr. Antibodies were diluted in a buffer consisting of 5% NDS and 0.4% Triton X-100 in PBS and incubated for 1–2 nights at room temperature. The following primary antibody concentrations were used: Mouse anti-CGRP (Abcam, 1:500–1:1,000); Rabbit anti-NPY (Cell Signaling, 1:500); Goat anti-ChAT (Millipore Sigma, 1:500); Rabbit anti-Ucn (Sigma-Aldrich, 1:500); Chicken anti-TH (Abcam, 1:1,000); Chicken anti-GFP (Aves, 1:2,000). Sections were rinsed 3 times with PBS, then incubated in secondary antibodies for 1.5–3 hr at room temperature. All secondary antibodies were used at 1:1,000 in 5% NDS and 0.4% Triton X-100 in PBS (Key Resources Table). Finally, sections were incubated in DAPI (Invitrogen, 1:10,000) for 10 minutes at room temperature, rinsed twice with PBS, and mounted with Vectashield hard-set medium (Vector Labs). Samples were imaged on either an Olympus VS120 fluorescent microscope or a Zeiss LSM 800 confocal microscope.

Cells were washed and lysed for 30 minutes at +4 °C in equal volumes of ice-cold RIPA lysis buffer (10 mM Tris pH 8, 1%Triton, 0,1% SDS, 0,1% Deoxycholate, 140 mM NaCl, 1 mM EDTA) and a cocktail of protease inhibitors (1 mM DTT, 1 mM PMSF, 1 Protease inhibitor tablet/10 mL Sigma, St. Louis, USA). Homogenates were incubated in ice for 20 minutes, then clarified by centrifugation at 14460 g for 15 minutes. Supernatants were boiled for 5 minutes in Laemmli sample buffer. Protein concentration was determined by Bradford and then separated by 15% SDS-PAGE electrophoresis and electrotransferred to polyvinylidene difluoride membranes. Incubation was performed overnight with the primary antibodies: Rabbit anti-Y1 and Rabbit anti-Y2 (1:500, Bioss Cat. N. bs-1070R and Cat. N. bs-0937R, respectively), Rabbit anti-Y5 (1:500, Abcam Cat. N. ab43824), Rabbit Anti-NPY (1:1000), phospho-ERK1/2 (1:200) and ERK (1:200; all Cell Signaling Technology, Cat. N. 11976 S, 1:1000, Cat. N. #4695, Cat. N. #9106, respectively) and a horseradish peroxidase-conjugated rabbit secondary antibody (Amersham mouse Cat. N. NA93IVS, Amersham rabbit NA934VS). The membranes were developed by enhanced chemiluminescence kit (ECL, GE Healthcare Bio-Science, Piscataway, NJ, USA). Acquisition and densitometry analysis was performed by ChemiDoc and Image Lab 5.2.1 (Biorad).