T1892

Thioflavin S (Ths) staining was used to label the Aβ plaques. Briefly, the brain sections were washed three times and then stained with Ths (0.05 mg/mL) (T1892, Sigma-Aldrich) in dark for 10 min and followed by two washes with 50% ethanol and PBS. The sections were mounted and imaged by using the confocal microscope.

Thioflavin S (ThS) staining was carried out as previously described^{22 (link)}. Briefly, ThS (T1892, Sigma‒Aldrich) was dissolved in 50% ethanol followed by dilution in H₂O to 1 mM. Free-floating brain sections were washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄) and mounted onto a glass slide. The brain sections were incubated with 1 mM ThS for 5 min. Stained sections were washed in 100%, 95%, and 50% ethanol for 30 s each and then rinsed with 1X PBS twice. The sections were dried and cover-slipped with anti-fade fluorescent mounting medium (S3023, DAKO, Carpinteria, CA, USA). ThS-stained plaques were photographed using an Olympus BX51 microscope equipped with a DP71 camera. The number of plaques and the size of the stained area of the plaques were analyzed using MetaMorph Microscopy Automation & Image Analysis software (Molecular Devices, Sunnyvale, CA, USA).

For immunofluorescence labeling, free-floating sections were probed using the following primary antibodies: goat anti-ChAT (1:400, AB144P, Millipore), mouse anti-parvalbumin (1:1000, MAB1572, Millipore), rabbit anti-calbindin (1:2000, CB38, Swant), mouse anti-Aβ (6E10, 1:500, Sig-39320, Convance), rabbit anti-GFAP (1:500, Z0334, Dako), rat anti-CD68 (FA-11, 1:500, MCA1957, AbD Serotec), and rabbit anti-HIF1α (1:200, NB100-479, Novus Biologicals) followed by the appropriate secondary antibody (1:1000, Life Technologies) or incubated with thioflavin S (0.1% in water, T1892, Sigma-Aldrich). Sections were mounted onto slides and coverslipped using fluorescence mounting medium (Dako).
For DAB (3,3′-diaminobenzidine) staining of cholinergic axons in the cortex, the biotinylated donkey secondary antibodies (1:1000, Jackson ImmunoResearch Laboratories) and ABC reagent (Vector Laboratories) were applied following the primary antibody incubation and followed by the nickel-intensified DAB reaction. Brain slices were mounted on slides and coverslipped with DPX mounting medium (Sigma-Aldrich).

Amyloid plaques were stained with thioflavin-S. The sections were incubated in 0.25% potassium permanganate solution for 20 min, rinsed in distilled water, and then incubated in a bleach solution (2% oxalic acid and 1% potassium metabisulfite) for 2 min to deparaffinization and hydration. After rinsing with distilled water, sections were transferred to the blocking solution (1% sodium hydroxide and 0.9% hydrogen peroxide) for 20 min, incubated in 0.25% acidic acid for 5 s, washed with distilled water, and stained for 5 min in 50% ethanol 0.0125% Thioflavin-S was added (excitation/emission wavelength: 390/428 nm; #T1892, Sigma-Aldrich, MA, USA). Next, the sections were washed with 50% ethanol, placed in distilled water, and then covered with glass lids using a mounting medium [20 (link)].