2.2 Immunostaining
Mice were anesthetized with pentobarbital sodium (60 mg/kg body weight,
intraperitoneal injection), followed by transcardial perfusion with
phosphate-buffered saline (0.01 M PBS, pH 7.4), and subsequently with
4% formaldehyde (PFA) prepared at 4°C to fix the tissues. The brain was
extracted and fixed in 4% PFA for an additional 12 hours, after which
it was transferred to 15% sucrose (in 0.01 M PBS, pH 7.4) for one day
and 30% sucrose for another two days at 4°C. Subsequently, the brain
was sectioned coronally into 40 μm slices using a cryostat microtome
(Leica CM 1900, Leica Biosystems, Wetzlar, Germany), and the slices were
collected in PBS.
The brain sections were permeabilized in a blocking solution (5% goat
serum, 0.5% Triton X-100, and 0.01% sodium azide in 0.01 M PBS) for 1
hour at room temperature (RT). The sections were then incubated
overnight at 4°C with primary antibodies against CB1Rs (1:5000,
sc-518035, Santa Cruz) or parvalbumin (1:500, 195002, Synaptic Systems).
All antibodies were prepared in the blocking solution.
After washing the sections three times for 10 minutes each in 0.01 M
PBS, they were incubated with secondary antibodies: goat anti-mouse
Alexa Fluor 488 (1:250, 33206ES60, Yeasen) or goat anti-rabbit Alexa
Fluor 594 (1:300, 33112ES60, Yeasen) for 2 hours at room temperature
(RT). The sections were then washed several times in PBS, mounted,
dried, and counterstained with 4’,6-diamidino-2-phenylindole (DAPI,
BMU107-CN, Abbkine) before being cover-slipped. For double-staining, the
sections were incubated with the individual primary antibodies and then
revealed with their respective secondary antibodies. All antibodies used
are listed in Table S1.
Fluorescent signals from the sections were captured using a confocal
laser scanning microscope (LSM980, Zeiss) with a Plan-Apochromat 20×
objective lens (effective NA = 0.8). The signal intensity of the
fluorescence was semi-quantitatively analyzed using ImageJ software
(NIH). The channels were separated individually, and the region of
interest (ROI) was outlined to exclude background noise. The threshold
for each image was adjusted to a default value, and an appropriate
algorithm was selected for analysis. Fluorescence signal from the
sections were captured using a confocal laser scanning microscope
(LSM980, Zeiss) with a unified numerical aperture of the lens (effective
NA=0.8, Plan-Apochromat 20×). The signal intensity of fluoresce was
semi-quantitatively analyzed by ImageJ software (NIH). Channels were
splitted individually and the region of interest was traced out to
eliminate background noise. The threshold for each image was adjusted to
a default value while a proper algorithm was selected.
After setting the measurements, the results of Integrated Density
(IntDen) and IntDen/Area would be provided automatically. The counting
of cells was performed by unbiased stereological methods referred to
(West, 1999; Zhang et al., 2017). Part of the images were merged for
analysis of co-localization of CB1Rs with PV.