Ab133477

After
completing behavior tests, mice from the control and MS groups (n = 6 per group, 7–8 weeks old) were anesthetized
with pentobarbital sodium. Subsequently, they were intracardially
perfused with sterile saline, followed by 4% paraformaldehyde in 0.1
M PBS (pH 7.4). The brain was postfixed in 4% paraformaldehyde overnight
and dehydrated through an ascending sucrose series, including 15 and
30% (w/v) sucrose in 0.1 M PBS at 4 °C overnight. Subsequently,
floating sections (25 μm) of the DRN were obtained and subjected
to immunohistochemistry staining using anti-GPR55
(Abcam, ab203663; 1:200) and anti-TPH2 (Abcam, ab133477;
1:200) antibodies. After washing three times, sections were incubated
with secondary antibodies diluted in PBST for 2 h at room temperature
in the dark. DAPI diluted in 0.1 mM PBS (1:1000) was applied to mark
cell nuclei. A confocal fluorescence microscopy (Olympus, Japan) was
employed to observe and acquire the images. Image analysis and process
were carried out using ImageJ (NIH) by researchers blinded to the
experiments.

Western blotting
was performed as previously described.^{68 (link)} After completing behavior tests, mice were euthanized in the control,
MS, and O-1602 treatment groups (n = 3 per group,
7–8 weeks old). Subsequently, their brains were removed and
sliced, and the DRN was carefully excised under a microscope. Total
protein was extracted from DRN samples using the M-PER protein extraction
buffer. Protein concentration was determined using a BCA kit. Equal
amounts of protein samples were used for western blotting analysis.
The primary antibodies used in this experiment were as follows: GPR55
(Abcam, ab203663; 1:1000), GluN2A (Abcam, ab124913; 1:1000), GluN2B
(Abcam, ab254356; 1:1000), p-CaMKIIα (Abcam,
ab171095; 1:1000), CaMKIIα (Abcam, ab52476; 1:1000), p-GluA1R Ser831 (Abcam, ab109464; 1:1000), p-GluA1R Ser45 (Abcam, ab76321; 1:1000), GluA1 (Abcam, ab31232; 1:1000),
and TPH2 (Abcam, ab133477; 1:1000). β-Actin (Sigma-Aldrich,
A5316; 1:10,000) was used as a loading control. After three washes
with TBST for 10 min, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies. Then, the signal of the
target protein was detected and digitized using the ECL solution and
the ImageJ program. The band intensity of each blot was quantified
as a ratio relative to β-Actin. And we normalized all the data
to obtain the same value in the control group.