Rabbit anti pv

Mice were euthanized by cervical dislocation and brains were quickly removed. The cortex was dissected in PBS on ice and stored at −80 °C until use. Cortices were homogenized using a Dounce tissue grinder set (Loose followed by Tight pestle, Sigma) in ice-cold 300 µl RIPA buffer supplemented with Complete EDTA-free Protease inhibitor (Roche). Lysates were incubated for 10 min at 4 °C and collected by centrifugation at 5000 × g for 10 min. Protein concentration was determined using the bicinchoninic acid kit (Pierce) and BSA as a standard. Equal amount of SDS sample buffer, Laemmli 2× concentrate (Sigma) was added to each lysate and boiled for 5 min. Samples were separated by 15% SDS-PAGE gel and transferred onto the 0.45 μm pore size Immobilone-FL Membrane (Millipore) for 1 h at 20 V. Membranes were blocked in 3% BSA (Applichem) in TBS-T (TBS plus 0.1% Tween 20) for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibody: mouse anti-GAPDH (1:5000, Abcam) or rabbit anti-PV (1:10000, Swant) diluted in blocking solution. After washing with TBS-T, membranes were incubated with secondary horse anti-mouse HRP-conjugated (1:20,000; Vector laboratories) or anti-rabbit HRP-conjugated (1:20,000; GE Healthcare) in blocking solution for 1 h at room temperature. Detection was performed using the ECL chemiluminescence reagent (Pierce) and X-ray films (AGFA).

Immunofluorescent labeling was performed on 25 µm cryo-sectioned tissue with the following antibodies: mouse anti-Kv3.1b 1:400 (NeuroMab cat. # 75-041); rabbit anti-phosphoS6^SER240/244 1:400 (Cell Signaling Technologies cat. # 5364); rabbit anti-PV 1:400 (Swant cat. # PV 27); rat anti-SST 1:200 (Millipore cat. # MAB354). The appropriate 488, 594 or 647 Alexa-conjugated secondary antibodies were from Thermo Fisher Scientific (1:300 dilution). Sections were cover-slipped with Vectashield (Vector labs).