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.