Mouse anti creb

Mice were anesthetized with isoflurane and perfused with 4% paraformaldehyde before being analyzed by single- or double-labeled immunohistochemistry. L3 and L4 DRG were removed, post-fixed, and dehydrated before frozen sectioning at 20 µm. After the sections were blocked for 1 h at room temperature in 0.01 M PBS containing 10% goat serum and 0.3% Triton X-100, they were incubated with the following one or two primary antibodies over one or two nights at 4℃. The antibodies include rabbit anti-G9a (1:20, Abcam), mouse anti-CREB (1:100, Cell Signaling, Danvers, MA), guinea pig anti-MOR (1:1,000, EMD Millipore, German), and rabbit anti-KOR (1:200, Novus Biologicals, Littleton, CO). The sections were then incubated with either goat anti-rabbit antibody conjugated to Cy3 (1:200, Jackson ImmunoResearch, West Grove, PA), or goat anti-mouse antibody conjugated to Cy2 (1:200, Jackson ImmunoResearch) or goat anti-guinea pig antibody conjugated to Cy3 (1:200, Jackson ImmunoResearch) for 2 h at room temperature. Control experiments included substitution of normal mouse or rabbit serum for the primary antiserum and omission of the primary antiserum. All immunofluorescence-labeled images were examined using a Leica DMI4000 fluorescence microscope and captured with a DFC365FX camera (Leica, Germany). Single- or double-labeled neurons were quantified manually or by using NIH Image J Software.