Amino silane coated slides

Three days after Aβ1-42 (0.5 μl/ 0.5mM) injection in the hippocampus (AP, -2.00 mm; ML, ±1.30 mm; DV, -2.10 to -1.60 mm), the mice were humanely euthanized, and the brains were cut into 20-μm sections. Sections with obvious needle track were mounted on amino silane coated slides (Solarbio, China). Neuronal apoptosis was analyzed using In Situ Cell Apoptosis Detection Kit V (POD) (Boster, China) testing protocol. Briefly, the sections were incubated with DIG-labeled UTP and terminal nucleic acid transferase (2 h, 37°C). After incubation with blocking solution (60 min), the slides were sequentially incubated with biotinylated mouse anti-DIG antibody (60 min, room temperature) and streptavidin-biotin-peroxidase complex (60 min, room temperature). Positive cells were selected as 3, 3-diaminobenzidine-stained and were counted and compared between wild-type and knockout mice.

Mature miR-124 was detected using a double-DIG-labeled miRCURY LNATM miRNA detection probe has-miR-124-3p (sequence/5DiGN/GGCATTCACCG CGTGCCTTA/3DiG-N/) (Qiagen, Germany) and scramble probe as sequence/5DiGN/ACACTCG/ IXNA_ C/ggC TTATTgCCg/3DiG-N (Sangon Biotech, China). The enhanced in situ hybridization procedure was performed according to the Enhanced Sensitive ISH Detection Kit I (Boster, Cat#: MK1030, China) protocol. Briefly, the paraformaldehyde-fixed frozen samples were cut into 20-μm thick sections and mounted on amino silane coated slides (Solarbio, China). Each slide was then washed with methyl alcohol with 0.3% hydrogen peroxide (30 min, room temperature). After incubation with pepsin (2 min, 37°C) and pre-hybridization (3h, 60°C), slides were hybridized with miRNA probe (20 nM, 60°C) overnight After a stringent washing, all sections were blocked using blocking solution (60 min, 37°C). Each section was then sequentially incubated with biotinylated mouse anti-DIG antibody (60 min, 37°C), streptavidin-biotin complex (60 min, room temperature), and biotinylated peroxidase (60 min, room temperature), with a 20-min wash in 0.5M PBS between each step in the sequence. Following staining with 3, 3-diaminobenzidine, the slides were dehydrated in a graded ethanol, cleared in xylene, and covered with coverslips, then coated with neutral resins.