Mouse anti pkc δ

Sufficient sections were taken to cover the ovBNST and CeA regions and were processed to detect of pERK and PKC-δ. Free-floating sections were rinsed in Trisbuffered saline (TBS: 0.25 M Tris, 0.5 M NaCl, 0.1 mM NaF, pH 7.6) three times for 10 min each, followed by 5 min in TBS containing 3% H₂O₂ and 10% methanol. After immersed in blocking buffer (0.2% Triton X-100 and 10% normal horse serum in TBS) for 1 h, sections were probed with rabbit anti-phospho-p44/42 MAPK (ERK1/2; 1:1,000; Cell Signaling Technology) and mouse anti-PKC-δ (1:1,000; BD Biosciences) diluted in blocking buffer at 4°C overnight. Next, after three washes in TBS for 10 min each, sections were incubated in blocking buffer containing donkey anti-rabbit Alexa Fluor 546 (1:1,000, Invitrogen), donkey anti-mouse Alexa Fluor 647 IgG (1:1,000, Invitrogen), and Nissl Green (1:1,000, Invitrogen) at 4°C overnight. Then they were washed in TBS for three times, mounted on slides (microscope plain slides, Thermo Scientific) and coverslipped in medium (0.17 mm thickness, Thermo Scientific; Fluoromount-G, SouthernBiotech). For lesion verification in Experiment 3, rabbit anti-GFAP (1:1,000, Sapphire Bioscience) was used as the primary antibody and donkey anti-rabbit Alexa Fluor 488 (1:1,000, Invitrogen) as the secondary antibody. Rest of the procedures were the same as described above.