Sc 189

Within 30 min after the experiment, rats were perfused with phosphate-buffered saline (PBS) then 4% paraformaldehyde in PBS (200 mL each), and decapitated. Electrode tips were marked with electrolytic lesions (0.8 mA, 0.8 s). Brains were removed from skull, post-fixed in 4% paraformaldehyde, and embedded in paraffin. By making lesions after perfusion, we could examine the mPFC, PV/MD, and CA1/sub for Zif268 immunohistochemistry upon coronal sectioning (8 μm) and bright-field microscopy. Separate coronal sections were Nissl-stained to check electrode positioning.
Published immunohistochemical protocols were used^{57 (link)}. Briefly, endogenous peroxidase was blocked with hydrogen peroxide in PBS (pH 7.4), followed by microwave antigenic retrieval in sodium citrate buffer (pH 6.0). Coronal sections were then incubated overnight in blocking buffer containing the polyclonal antibody against Zif268/Egr-1 (sc-189, Santa Cruz Biotechnology; 1:100 dilution). Primary antibodies were detected with the biotinylated anti-rabbit IgG (E0353, Dako; 1:100 dilution) followed by the HRP Kit (PK6100, Vector Laboratories), and finally revealed with diaminobenzidine.

RIPA buffer (radioimmunoprecipitation assay buffer, pH 7.4) supplemented with protease inhibitors (Complete protease inhibitor cocktail, Roche Applied Science) was used for protein extraction from hippocampal tissue (n = 3–6 animals per group) followed by measuring protein concentrations using Bradford assays (Protein Assay Dye Reagent Concentrate, Biorad, Germany). After protein separation on 12% Bis-Tris gels, nitrocellulose membranes were used for protein blotting for 1.5 h at 4°C. Subsequently, membranes were blocked with Odyssey blocking solution (LI-COR Biosciences) and incubated with primary antibodies at 4°C overnight (anti-human alpha-synuclein, 1:100, 15G7, Enzo Life Sciences), (anti-GFAP, 1:100, DAKO), (anti-NURR1, 1:250, sc-991, Santa Cruz), (anti-EGR1, 1:250, sc-189, Santa Cruz), (anti-synaptophysin, 1:2000, MAB368, Millipore), (anti-synapsin, 1:500, cs-2312, Cell Signaling), (anti-VAMP-1/2/3, 1:100, sc-133129, Santa Cruz), (anti-complexin 1/2, 1:250, sc-33603, Santa Cruz), (anti-PSD95, 1:1,000, ab12093, Abcam). Incubation with respective near-infrared fluorescent secondary antibody (IRDye 800CW or IRDye 680LT, LI-COR Bad Homburg, Germany) was followed by membrane detection and image quantification using LI-COR Odyssey imaging system and Image Studio, respectively.