Rabbit anti pv

The protein expression of PV was measured using Western blot. The vHipp was dissected from a subset of control and FAB rats (n = 5 per group) and homogenized in ice-cold buffer (750 mL) containing a protease inhibitor. Samples were centrifuged (14,000 rpm for 2 min), and the supernatant, containing protein fractions, was collected. Protein concentrations were determined using the Bradford method prior to incubation with Laemmli sample buffer containing 5% dithiothreitol (10 min at 90 °C), then separated (20 µg/lane) at 200 mA on Any kD™ Mini-PROTEAN^® TGX™ Precast Protein Gels. Proteins were transferred (100 mA for 1 h) to a nitrocellulose membrane. The membranes were then washed 3 times (10 min each) in TBST and blocked (30 min in 5% BSA or milk in TBST) before incubation in rabbit anti-PV (1:5000; Abcam, Waltham, MA, USA) or mouse anti-GAPDH (1:1000; Abcam) antibody for 1 h. Membranes were washed 3 times (10 min in TBST), followed by either HRP-anti-rabbit 1:10,000 or HRP-anti-mouse 1:5000 (1 h at room temperature). Next, membranes were treated with Pierce™ ECL Western Blotting substrate (1 min) and exposed to high-performance chemiluminescence film (Amersham Hyperfilm™ ECL). Western blot films were scanned, and optical density was measured using ImageJ and analyzed by the Mann–Whitney rank sum test.