Frozen slicer

Larvae at 3rd day of the fifth instar were fed with mulberry leaves that were smeared with FITC-dextran of different molecular masses (Sigma-Aldrich Corp., Saint Louis, MO, USA). The anterior midgut was dissected and fixed in 4% paraformaldehyde overnight at 4 °C. Tissues were embedded in OCT (Cell Path, UK) and sectioned using a frozen slicer (Thermo Scientific Inc., Waltham, MA, USA) at −24 °C. Transverse 20-µm sections were analyzed using fluorescence microscopy (Olympus, Tokyo, Japan).

After the behavioural tests, the mice were anaesthetized and transcardially perfused with saline. Then, the brains were removed, and the hemispheres were separated. Brain samples assigned for immunohistochemical staining were immersed in OCT at -80 °C before coronal sectioning on a Frozen slicer (Thermo Fisher Scientific, USA) (10 μm thickness). The sections were restored to room temperature, fixed with cold acetone for 5 min, and permeabilizing agent was used for 5 min. Then, the cells were rinsed three times at room temperature for 5 min each with 1x PBS (pH 7.2-7.4) and blocked with 10% bovine serum in 0.1% Triton X-100 for 30 min. The blocking solution covered all brain tissues and prevented the tissue from drying out. Then, the samples were incubated with primary antibodies overnight at 4 °C. The primary antibodies included rabbit anti-NeuN (1:500, Abcam), rabbit anti-GFAP (1:200, CST), rabbit anti-Iba-1 (1:100, Abcam), rabbit anti-CD68 (1:300, Abcam), rabbit anti-APP (1:100, Millipore), and mouse anti-β-amyloid specific for Aβ₄₂ (1:200, CST). The next morning, the sections were incubated for 1 h at room temperature with DyLight 488-/555-conjugated goat anti-rabbit/mouse IgG (1:200, Abcam) and stained with DAPI for 3 min. Fluorescence images were captured using an Olympus FV3000 confocal microscope.