2.6 Western blot
Western blot analysis was performed as required by BJP guidelines(Alexander, Roberts et al., 2018). After the rats were decapitated, the brains were immediately removed to a prechilled brain matrix with a 1.0 mm coronal slice thickness (RWD Life Technology, Shenzhen, China). PrL and IL tissues were harvested on ice guided by the Paxinos and Watson rat brain atlas(Paxinos & Watson, 1986) and stored separately in prechilled microcentrifuge tubes at −80°C until assayed. The tissue was homogenized in RIPA buffer (Solarbio, Cat# R0010, China) supplemented with protease inhibitors (Solarbio, Cat# P0100, China) and phosphatase inhibitors (Solarbio, Cat# P1260, China). Protein (45 μg) was separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, USA). The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies, including anti-β-actin (1:4000; Rabbit mAb, ABclonal, Cat# AC038, Wuhan, China), anti-β-Amyloid (Aβ) (1:50; Mouse mAb, Santa Cruz Biotechnology, Cat# sc-28365, RRID:AB_626669), anti-Tau (1:1000; Mouse mAb, Cell Signaling Technology, Cat# 4019, RRID:AB_10695394), anti-Phospho-Tau (Ser199) (1:1000; Mouse mAb, Cell Signaling Technology, Cat# 29957, RRID:AB_2798984), anti-glial fibrillary acidic protein (GFAP) (1:1000; Rabbit mAb, Cell Signaling Technology, Cat# 80788, RRID:AB_2799963), anti-glutamate-aspartate transporter (GLAST) (EAAT-1 in humans, 1:1000; Rabbit mAb, Cell Signaling Technology, Cat# 5684, RRID:AB_10695722), anti-glutamate transporter-1 (GLT-1) (EAAT-2 in human, 1:500; Rabbit mAb, Abcam, Cat# ab41621, RRID:AB_941782), anti-ionized calcium-binding adaptor molecule-1 (Iba1) (1:1000; Rabbit mAb, Cell Signaling Technology, Cat# 17198, RRID:AB_2820254) and anti-BDNF (1:500; Rabbit mAb, Abcam, Cat# ab108319, RRID:AB_10862052) in TBST buffer (Tris-buffered saline + 0.1% Tween-20) overnight at 4°C. The blots were then washed with TBST three times before incubation with HRP goat anti-mouse IgG (H+L) antibody (1:2000; ABclonal, Cat# AS003, Wuhan, China) or HRP goat anti-rabbit IgG (H+L) antibody (1:2000; ABclonal, Cat# AS014, Wuhan, China) for 2 h at room temperature. After 3 × 5 min TBST washes, an ECL Enhanced Kit (ABclonal, Cat# RM00021, Wuhan, China) was used for detection enhancement, and blots were visualized using ImageJ software. The results were normalized to the protein expression level of β-actin.