Irdye 680lt donkey anti rabbit igg secondary antibodies

Total proteins were extracted as previously described.²³, ²⁶, ²⁷ The tissues were briefly homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Biotime) supplemented with 1% phenylmethylsulfonyl fluoride (Sangon Biotech) and 1× PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science). Protein concentration was measured using a butyleyanoacrylate (BCA) kit according to the manufacturer's instructions (Thermo Scientific). The proteins were separated using SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% bovine albumin (BSA) with a TBST buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), followed by incubation with primary antibodies diluted in TBST containing 2.5% BSA at 4°C overnight: anti‐PPM1F (1:500, PA5‐15571, Invitrogen), anti‐p‐AMPK antibody (1:1000, #2535, Cell Signaling Technology), anti‐AMPK antibody (1:1000, ab32047, Abcam), and anti‐β‐actin antibody (1:1000, 4970, Cell Signaling Technology). Then, the membranes were washed with 1× TBST and incubated with IRDye 680LT donkey anti‐rabbit IgG secondary antibodies (1:5000, 926–68,023, Li‐COR Biosciences). Fluorescence was visualized and analyzed using an Odyssey infrared imaging system (Li‐COR Biosciences).

The dissected brain tissue samples (mPFC, hippocampus and DRN) were homogenized in a lysis buffer containing 50 mM Tris-HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), phenylmethylsulfonyl fluoride, and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany). The denatured protein was separated on a SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. The membrane was blocked in a solution of tris-buffered saline with 1% dried milk and 0.1% tween 20, and then incubated with the following primary antibodies diluted in a blocking solution: anti-TPH2 antibody (1:1000; ab184505, Abcam, Cambridge, UK), anti-SERT antibody (1:1000; ab172884, Abcam, Cambridge, UK), anti-β-actin antibody (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IRDye 680LT donkey anti-rabbit IgG secondary antibodies (1:5000; 926-68023, Li-COR Biosciences, Lincoln, NE, USA). The fluorescence was visualized and analyzed using an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA).