Hm650v microtome

Mice were transcardially perfused with ice-cold PBS to remove blood. Brains were extracted, and the right cerebral hemisphere was fixed by immersion in a 4 % paraformaldehyde solution for 24 h at 4 °C. Forty micrometer-sagittal sections were cut on a vibrating HM650V microtome (Thermo Scientific) and were preserved in PBS/azide 0.1 %. Staining with thioflavin-S (ThioS; Sigma-Aldrich), a specific β-sheet strand intercalant, was performed on brain sections as described previously [21 (link)]. Image acquisition was performed using a digital inverted fluorescence microscope (EVOS-xl; Life Technologies) with a ×4 lens. Plaques were quantified using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA) by measuring the area of ThioS staining in a well-defined selected area of the hippocampus.

For immunohistological analysis, free-floating coronal sections (50 μm) were generated from agarose-embedded fixed brains using a vibrating HM650V microtome (ThermoFisher), and were preserved in PBS-sodium azide 0.02% at 4 °C. Prior to immunomarking, sections were washed in PBS and subsequently blocked and permeabilized with PBS-BSA 3%-TritonX100 0.5% for 1 h at rt. Sections were then incubated with anti-human Aβ clone W0-2 (1:100) overnight at 4 °C as a marker for Aβ-containing species. After three PBS washes and incubation with goat anti-mouse IgG Alexa Fluor™ 647 secondary antibody (1:500) for 1 h at rt, slices were finally washed three times with PBS and mounted on SuperFrost® slides. Slides were then incubated with ThT (0.1 mg/ml in ethanol 50%) for 15 min at rt as a marker for fibrillar deposits. After three washes with ethanol 80% and a final wash with ultrapure water, coverslips were mounted with Mowiol® 4–88-glycerol. W0-2 and ThT staining were detected with standard FITC/Cy5 and GFP filters, respectively, at an EVOS® FL Autofluorescence microscope. Counting of double-positive dots was performed on ImageJ.

Directly after dissection, brains were trimmed and placed in ice cold artificial cerebrospinal fluid (aCSF) containing (in mM): 130 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2 CaCl₂, 1 MgCl₂, and 10 glucose; pH 7.4, bubbled with carbogen (95% O₂, 5% CO₂). They were cut into 250 μm thick parasaggital slices containing the neocortex and hippocampus using a vibratome (HM650V, Microtome, Thermo Fisher Scientific, Waltham, MA, USA). Slices were washed five times with acidified Hank's Balanced Salt Solution (Sigma-Aldrich, Munich, Germany), transferred onto Biopore membranes (Millicell standing insert, Merck Millipore, Burlington, VT, USA) and kept in an incubator at the interface between culture medium and humidified air containing 5% CO₂ until used for experiments (Stoppini et al., 1991 (link)).
Experiments were performed in layers II/III of the somatosensory cortex and the CA1 region of the hippocampus at room temperature. During experiments, slices were constantly superfused with aCSF at a prefusion speed of 2–2.5 ml/min. All chemicals were purchased from Merck/Sigma-Aldrich (St. Louis, MO, USA) or AppliChem (Darmstadt, Germany) except for (2R)-amino-5-phosphonovaleric acid (APV), which was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).