2.7 Tissue preparation and immunofluorescence staining
The immunohistochemistry procedures followed the recommendations by the
British Journal of Pharmacology (Alexander,
Roberts, Broughton, Sobey, George, Stanford et al., 2018). Rats from
each group (n=7) were deeply anaesthetised and transcardially perfused
with 0.9% saline, followed by 4% paraformaldehyde solution. The brains
were fixed in 4% paraformaldehyde, transferred to 30% sucrose solution
for 5 days, and embedded in OCT at -20℃. Serial coronal sections (30 μm)
were obtained using a cryostat microtome (Leica, Wetzlar, Germany).
Brain slices were washed three times with phosphate-buffered saline
(PBS), permeabilised with 0.1% Triton X-100 for 10 min, blocked in 10%
goat serum (Invitrogen Corp. USA, Cat#
16210064)
for 1 h at room temperature, and incubated with the primary antibody
overnight at 4°C. For the detection of BrdU-labelled nuclei, the
specimens were previously incubated in 2N HCl at 37℃, followed by a
10-min rinse in 0.1 M sodium tetraborate pH 8.5 and two rinses with PBS
before the blocking step. The following primary antibodies were diluted
in blocking solutions: mouse monoclonal IgG anti-BrdU (Biolegend, USA;
1:250, Cat# 339802, RRID: AB_1595495), rabbit monoclonal IgG anti-NeuN
(Cell Signaling Technology, USA; 1:500, Cat# 24307, RRID: AB_2651140),
rabbit monoclonal IgG anti-doublecortin (DCX, Cell Signaling Technology,
USA; 1:500, Cat# 4604, RRID: AB_561007), mouse monoclonal IgG
anti-glial fibrillary acidic protein (GFAP, Sigma, USA; 1:500, Cat#
G3893, RRID: AB_477010), and rabbit polyclonal IgG anti-Iba1
(Proteintech, China; 1:500, Cat# 10904-1-AP, RRID: AB_2224377). The
brain slices were washed with PBS before incubation with Alexa
Flour-conjugated secondary antibodies (Alexa Fluor 555-conjugated donkey
anti-mouse IgG, Cat# A-31572, RRID: AB_162543; Alexa Fluor
488-conjugated donkey anti-rabbit IgG, Invitrogen Corp. USA; 1:500,
Cat# A-21202, RRID: AB_141607) and DAPI (4ʹ,
6-diamidino-2-phenylindole, Invitrogen Corp. USA; 1:1000; RRID:
AB_2307445) for 1 h at room temperature. Finally, the sample slices
were mounted on glass slides using a fluorescence mounting medium (DAKO,
Danmark, Cat# S3023, RRID: SCR_013530).