Western Blot Analysis of PV and GAD67 in FST Mice

Western Blot analysis was conducted in VEH/KET mice. Brains were collected 4 h after the FST, and immediately frozen on dry ice and stored at −80 °C. We chose a 4-h time point post-FST to avoid for potential confound due to molecular changes induced by acute exposure to FST. The PFC was dissected on dry ice in a cold room maintained at −15 °C and then processed as described previously^{22 (link)}. The following antibodies were used: anti-parvalbumin (rabbit anti-PV, 1:500—ABCam, MA, USA) and anti-GAD67 (rabbit anti-GAD67 1:1000—Thermo Fischer Scientific, MA, USA). Signal was visualized using Clarity Western ECL Substrates with an HRP-conjugated secondary antibody for digital imaging (BioRad, CA, USA). Signal intensity was measured using the Image J Studio software whereby the mean gray value of each band of interest was measured and subtracted from the background value. The ratio of each protein of interest band value over the loading control (β-actin) value was then calculated.