Anti fading mounting media

One hour after the end of the final behavioral test, rats were anesthetized with sodium pentobarbital (450 mg/Kg, i.p.) and then perfused transcardially with 250 ml of 0.9% saline followed by 500 ml of cold fresh 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PBS) at pH 7.4. Brains were removed and fixed overnight in 4% PFA, and transferred to 30% sucrose in 0.1 M PBS for 48 h, for cryoprotection. Frozen sections were cut coronally (40 μm) with a cryostat (CM 1850; Leica) at different levels of the prefrontal cortex, paraventricular thalamus, and amygdala.
Sections were initially blocked in a solution of 2% normal goat serum (NGS, Vector Laboratories, United States) and 0.1% Tween (Tween-20, Sigma-Aldrich, United States) in 0.1 M PBS (pH 7.4) for 1 h. Afterward, sections were incubated overnight at room temperature with anti-c-Fos serum raised in rabbit (Ab-5, Oncogene Science, United States) at a concentration of 1:2,000. Sections were then incubated for 2 h at room temperature in a solution of fluorescent secondary-antibody Alexa Fluor 488 (1:500; Life Technologies). Sections were cover slipped with anti-fading mounting media (Vector Laboratories) and examined under an epifluorescent microscope.

The paraffin sections were baked at 65 °C overnight. Slides were then deparaffinized and rehydrated. Dako endogenous blocking reagent (S2003, Dako, Carpinteria, CA, USA) was then used to quench endogenous peroxidase for 15 min. Non-specific binding sites were blocked with 1:10 normal horse/goat serum (S-2000, Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Primary antibodies: 1:400 dilution of MMP13 (ab39012, Abcam, Cambridge, UK), 1:1 000 dilution of ColX (ab49945, Abcam, Cambridge, UK); 1:400 dilution of Admts4 (ABT178, Millipore, Billerica, MA), and 1:500 dilution of Adamts5 (ab41037 Abcam, Cambridge, UK) were added, and the slides were incubated at 4 °C overnight. For IHC assays, the secondary biotinylated goat anti-mouse antibody (BA-9200, Vector Laboratories) at the dilution of 1:200 was added for 30 min on the second day, followed by incubation with 1:250 streptavidin (21130, Pierce, Rockford, IL, USA) for 30 min. Positive staining was detected by Romulin AEC Chromagen (Biocare Medical RAEC810L, Concord, CA, USA). For IF staining, an appropriate secondary antibody conjugated to a fluorescence probe was added, incubated at room temperature for 1 h, rinsed in PBS, and mounted in an anti-fading mounting media (Vector Laboratories, Burlingame, CA). Results were obtained using an Olympus BX43 upright microscope (Olympus Optical, Tokyo, Japan).