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 used57 (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.
Published immunohistochemical protocols were used57 (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.
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