In situ cell apoptosis detection kit 5 pod

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.

After animals were anesthetized with pentobarbital (100 mg/kg intraperitoneally), they were sacrificed by transcardiac perfusion by injecting them with 0.9%, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains of rats from each group were removed, embedded in paraffin, and sectioned into 4 μm coronal sections on a sliding microtome. The sections were stained with Nissl, and images were captured using a digital camera. TUNEL staining was performed to evaluate hippocampal neuronal cell apoptosis. For TUNEL staining, sections were boiled by microwaving in a citrate buffer (10 mM, pH 6.4) for 5 min for antigen retrieval after deparaffinization and rehydration. Then, the sections were directly incubated with TUNEL mix from the in situ Cell Apoptosis Detection Kit V (POD) (Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer's protocol. Six sequential slices of the hippocampus were used with a 5 μm interval between each two adjacent sections from each animal group to assess the number of pyramidal neurons in the CA1 region of the hippocampus.