2.3 Western Blot analysis
The Cnr1flox/flox and PV-Cre;Cnr1flox/flox transgenic mice were sacrificed and used for the immunoblot after anesthesia. Brain tissues were extracted and lysed using homogenizer in ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor AEBSF and reductant β-ME. The lysate was centrifuged twice at 15,000×g for 20 min at 4 ℃ to remove insoluble debris, and the supernatant was transferred to a sterile EP-tube for another 20 min centrifugation. The concentration of total proteins in solubilized fractions was determined by Bradford assay using spectrophotometer at 595 nm. Then the protein samples were mixed with 5×Loading buffer (Dithiothreitol contained) and denatured in Block Heater for separation by 15% SDS-PAGE (Tanon Electrophoresis System). Separated proteins were transferred onto 0.22 μm PVDF membranes individually, and the membranes were blocked in TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20) with 5% non-fat milk contained for 1 hour at RT. The primary antibodies used for target and reference proteins were anti-CB1Rs (1:5000, sc-518035, Santa Cruz) and anti-GAPDH (1:5000, AF7021, Affinity) respectively, and they were further detected with HRP-linked secondary antibodies for ECL assessment (Table S1). The blotting bands were visualized with chemiluminescence imaging system (JS-1070P, Peiqing). Band intensities were quantified through ImageJ software (NIH) to analyze the protein expression of CB1Rs relative to GAPDH in the same sample.
2.4 RT-qPCR
Brain tissues isolated from mice were homogenized in Trizol, and then mixed with chloroform for 2 min of shaking and 3 min of quiescence at Room Temperature to extract total RNA. The mixture was centrifuged at 12,000×g for 15 min at 4 ℃ to separate water-organic phases. Then the water phase was preserved with isopropyl alcohol added and mixed well before precipitation at -80 °C for 2 hrs. The new mixture was centrifuged for another 15 min at 12,000×g to precipitate RNA, and then washed by ice-cold 80% ethanol. Finally, the gelatinoid RNA precipitant was desiccated and dissolved in 0.1% DEPC ddH2O. Concentration measurement by OneDrop spectrophotometry (OD1000+, WINS) combined with integrity detection through agarose gel electrophoresis were conducted to evaluate the quality of RNA.
Reverse-transcription and quantitative real-time PCR were subsequently performed using the cDNA Synthesis Kit (11141ES60, YEASEN) and qPCR Kit (11184ES08, YEASEN) respectively. Data from qPCR were normalized with the method of 2−ΔΔCt, and the relative transcription of Cnr1 was derived from the transcript ratio of cnr1 toβ-actin , the reference gene. All the regents and materials used were RNase-free, and the primers used to amplify the target genes are shown in Table S2.