L1516 2mg

Free-floating sections were rinsed 7 times for 15 min each with 1x tris-PBS (TPBS; Tris–HCl 10 mM, sodium phosphate buffer 10 mM, 0.9% NaCl, pH 7.4), and incubated with the following primary antibodies: (1) rabbit polyclonal anti-c-Fos antibody (RPCA-c-Fos-AP, Encorbio, USA), diluted 1:500; (2) Wisteria floribunda agglutinin (WFA; L1516–2MG, Sigma Aldrich, USA), diluted 1:200; (3) mouse monoclonal anti-PV antibody (235, Swan, Swiss antibodies, Switzerland), diluted 1:1000; or (5) mouse monoclonal anti-CB antibody (300, Swan, Swiss antibodies, Switzerland) diluted 1:1500; (6) mouse monoclonal anti-CaMKII antibody (MA 1–048, Thermo Scientific, USA) diluted 1:50. Incubations were at 4º C for 48 hr in TPBS 0.1M Triton X-100 containing 3% of donkey serum (Santa Cruz Biotechnology sc-2044). After three 15-min TPBS rinses, tissue was incubated for 2 hr at room temperature protected from light with one of the following secondary antibodies with conjugated fluorochromes: (1) Alexa Fluor 488 donkey anti-rabbit (A-21206, Life Technologies, USA), diluted 1:500; (2) Alexa Fluor 647 donkey anti-mouse (715–605-150, Jackson Labs, USA), diluted 1:500; or (3) biotinylated goat anti-rabbit conjugated with streptavidin Texas red (Vector Labs, UK), diluted 1:500. Once the fluorescence reaction occurred, sections were mounted using Mowiol 4–88 reagent (475904–100GM, EMD Millipore, USA).