Aβ1 42

The paraffin sections were dewaxed and hydrated before H&E staining. After dewaxing and hydration, H&E staining was performed and eventually observed under a light microscope. For immunohistochemistry, the sections were incubated in 3% H₂O₂ for 15 min and 80% carbinol for 30 min, and then they were incubated in blocking solution for 1 hr at 37?. Subsequently, the sections were incubated at 4? overnight with the following primary antibodies: ATG5 (1:200, Bioworld Technology, Shanghai, China), ATG7 (1:200, Bioworld Technology), and Aβ_1-42 (1:400, Abcam). After washing with PBS three times, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hr at 37?, and then they were treated with 3, 3-diaminobenzidine (DAB). The results were imaged using a Nikon ECLPSE 80i (Nikon, Tokyo, Japan).

Aβ_1–42 (Abcam, Cambridge, UK) was dissolved in sterile phosphate buffer (PBS; 0.01 M NaH₂PO₄, 0.15 M NaCl, pH 7.4) at a concentration of 1 mg/mL and stored at −20 °C. Before in vivo injection, aliquots of Aβ peptide were aggregated by incubation at 37 °C for 72 h [24 (link)].
Human brain material was provided via the rapid autopsy program of the Netherlands Brain Bank (NBB), which provides post-mortem specimens from clinically well documented and neuropathologically confirmed cases.

Anti‐miR‐140‐5p (AM‐140) was synthesized by Metabion (Martinsried, Germany). AM‐140 contained 2’‐O‐methyl modifications (5′‐(2’OMe‐C) *(2’OMe‐U) *a* cca uag ggu aaa acc *(2’OMe‐A)* (2’OMe‐C)*(2’OMe‐U) *(2’OMe‐G)‐3′). Aβ (1–42) (CAS number: ab120301) were purchased from Abcam. Oligomeric Aβ1–42 was prepared as originally established by Klein et al.²⁴ with a slight modification. The lyophilized synthetic human Aβ1–42 peptides were dissolved in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (Sigma‐Aldrich, 105,228) and then evaporated overnight at room temperature in aliquots. The resulting Aβ peptide film was stored at −80°C. 24 h before use, the films were resuspended in anhydrous dimethyl sulfoxide (DMSO) to 5 mM, and then diluted in phosphate‐buffered saline (PBS; pH 7.4) to a concentration of 100 μM and stored at 4°C.

For H&E staining, the 5 μm sections were dewaxed and hydrated, then stained with hematoxylin and eosin solutions, and observed under light microscope. For Nissl staining, tissue sections were stained with cresol violet and Nissl differentiation solutions according to the instructions (Beyotime), and observed under light microscope. For Immunohistochemistry staining, the sections were incubated with 3% H₂O₂ for 15 min, and then in blocking solution for 45 min. Subsequently, the sections were incubated at 4 °C overnight with the following primary antibody: Aβ_1–42 (1:400, Abcam). After washing in PBS for 3 times, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 4 h at 37 °C. Then, the sections were reacted with 3, 3-diaminobenzidine (DAB), and imaged using a Nikon ECLPSE 80i (Nikon, Tokyo, Japan).

To induce Alzheimer's disease in the present study, beta-amyloid 1–42 (Aβ_1-42) was injected into the CA1 region of the dorsal hippocampus of Aβ rats using a stereotaxic device (RWD, China)^{12 (link)}. To prepare the injection solution, 1 mg/mL of Aβ_1-42 (Abcam, Cambridge, UK, Cat no. ab120301) was initially diluted with ice-cold 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and incubated at room temperature for at least 60 min with occasional vortexing. Next, the HFIP was removed using a SpeedVac, and the peptide film was stored at − 80 °C. Then for aggregation, the peptide was first resuspended in DMSO (5 mM), sterile phosphate buffer saline (PBS) was added to bring the peptide to a final concentration of 1 μg/μL, and the Aβ_1-42 peptide was incubated at 37 °C for 72 h. To perform stereotaxic surgery, the animals were anesthetized with isoflurane inhalation. Aβ_1-42 suspension (1 μl/site) was injected into the CA1 region of the dorsal hippocampus of A-C, and A-Ex groups (A-4.2, L ± 3.0, V-2.0 mm) based on the Paxinos and Watson atlas by a Hamilton syringe attached to an infusion pump^{12 (link)}. Moreover, isotonic saline solution was injected into the sham animals. To confirm Alzheimer’s induction, pathological slides were prepared from the hippocampus of three animals/per group 10 days after surgery using thioflavin immunostaining of beta-amyloid plaques.