An 8-month-old 5XFAD mouse was anesthetized with Tribromoethanol
(Avertin) 125–250mg/kg IP and transcardially perfused with saline. Brain
was post-fixed with 4% PFA in PBST (0.1% Tween (0777, VWR) in 1×PBS) for
24 h. Tissue was then transferred to 30% sucrose (57-50-1, Fisher Chemical).
Tissue was embedded in O.C.T. compound (23-730-571, Fisher Healthcare) and
hemibrain was sagitally sectioned on a cryostat (CM1950, Leica) at 30 μm
thick. Sections were stored at −20 °C in cryoprotectant (30%
glycerol (G5516, Sigma-Aldrich) and 30% ethylene glycol (293237, Sigma-Aldrich)
in 1×PBS). Prior to staining, sections were washed three times in PBST
for 10 min each time and then treated for antigen retrieval with 10 mM sodium
citrate (S279–500, Fisher Chemical) and 0.5% Tween in PBST at 85
°C for 10 min. Sections were blocked in normal donkey serum (D9663,
Sigma-Aldrich) for 1 h and incubated with anti-β-amyloid antibody (2454,
Cell Signaling Technology) at 4 °C overnight. Following three PBST
washes, sections were then stained with donkey anti-rabbit Alexa Fluor 647
conjugated antibody (A31573, Invitrogen) at RT for 1 h. Both primary and
secondary antibodies were diluted to 1:1000 in blocking buffer. Nuclei were
stained with DAPI (10236276001, Sigma-Aldrich) diluted to 1:10,000 in PBST at RT
for 2 min. Sections were then wet mounted onto coverslips and dried at 4
°C overnight before imaging.
(Avertin) 125–250mg/kg IP and transcardially perfused with saline. Brain
was post-fixed with 4% PFA in PBST (0.1% Tween (0777, VWR) in 1×PBS) for
24 h. Tissue was then transferred to 30% sucrose (57-50-1, Fisher Chemical).
Tissue was embedded in O.C.T. compound (23-730-571, Fisher Healthcare) and
hemibrain was sagitally sectioned on a cryostat (CM1950, Leica) at 30 μm
thick. Sections were stored at −20 °C in cryoprotectant (30%
glycerol (G5516, Sigma-Aldrich) and 30% ethylene glycol (293237, Sigma-Aldrich)
in 1×PBS). Prior to staining, sections were washed three times in PBST
for 10 min each time and then treated for antigen retrieval with 10 mM sodium
citrate (S279–500, Fisher Chemical) and 0.5% Tween in PBST at 85
°C for 10 min. Sections were blocked in normal donkey serum (D9663,
Sigma-Aldrich) for 1 h and incubated with anti-β-amyloid antibody (2454,
Cell Signaling Technology) at 4 °C overnight. Following three PBST
washes, sections were then stained with donkey anti-rabbit Alexa Fluor 647
conjugated antibody (A31573, Invitrogen) at RT for 1 h. Both primary and
secondary antibodies were diluted to 1:1000 in blocking buffer. Nuclei were
stained with DAPI (10236276001, Sigma-Aldrich) diluted to 1:10,000 in PBST at RT
for 2 min. Sections were then wet mounted onto coverslips and dried at 4
°C overnight before imaging.